Method to trigger rna interference

ABSTRACT

A method to generate siRNAs in vivo is described, as are constructs and compositions useful in the method. The method does not depend on the use of DNA or synthetic constructs that contain inverted duplications or dual promoters so as to form perfect or largely double-stranded RNA. Rather, the method depends on constructs that yield single-stranded RNA transcripts, and exploits endogenous or in vivo-produced miRNAs or siRNAs to initiate production of siRNAs. The miRNAs or siRNAs guide cleavage of the transcript and set the register for production of siRNAs (usually 21 nucleotides in length) encoded adjacent to the initiation cleavage site within the construct. The method results in specific formation of siRNAs of predictable size and register (phase) relative to the initiation cleavage site. The method can be used to produce specific siRNAs in vivo for inactivation or suppression of one or more target genes or other entities, such as pathogens.

REFERENCE TO RELATED APPLICATIONS

This a division of co-pending U.S. patent application Ser. No.13/827,450, filed Mar. 14, 2013; which is a continuation of U.S. patentapplication Ser. No. 13/216,942, filed Aug. 24, 2011 and issued as U.S.Pat. No. 8,476,422 on Jul. 2, 2013; which is a continuation of U.S.patent application Ser. No. 11/334,776, filed Jan. 6, 2006 and issued asU.S. Pat. No. 8,030,473 on Oct. 4, 2011; which claims the benefit ofU.S. provisional application No. 60/642,126, filed Jan. 7, 2005. Each ofthese prior applications is incorporated herein by reference in itsentirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with United States government support pursuantto grant MCB-0209836 from the National Science Foundation, grant A143288from the National Institutes of Health, and grant 2005-35319-15280 fromthe USDA; the United States government has certain rights in theinvention.

FIELD

This disclosure relates to methods of regulating gene expression in vivoin plant, fungi, and invertebrate cells, as well as constructs andcompositions useful in such methods. Further, it relates toRNAi-inducing nucleic acid constructs having a microRNA or siRNA targetsequence (initiator sequence) and one or more siRNA-generating sequencesdirected to one or more target genes or RNAs, whereby thesiRNA-generating sequences are in 21-nucleotide register with thecleavage site guided by the microRNA or siRNA initiator.

BACKGROUND

Mechanisms that suppress the expression of specific cellular genes,viruses or mobile genetic elements (such as transposons andretroelements) are critical for normal cellular function in a variety ofeukaryotes. A number of related processes, discovered independently inplants (Matzke et al., Curr. Opin. Genet. Dev. 11:221-227, 2001),animals (Fire et al., Nature, 391:806-811, 1998) and fungi (Cogoni,Annu. Rev. Microbiol. 55:381-406, 2001), result in the RNA-directedinhibition of gene expression (also known as RNA silencing). Each ofthese processes is triggered by molecules containing double-stranded RNA(dsRNA) structure, such as transcripts containing inverted repeats ordouble-stranded RNA intermediates formed during RNA virus replication.Non-dsRNAs, also referred to as aberrant RNAs, may also function asinitiators of RNA silencing. Such aberrant RNAs may be converted intodsRNAs by silencing-associated RNA-dependent RNA polymerases (RDRs),which have been identified in plants, fungi and C. elegans (Tuschl,ChemBiochem, 2:239-245, 2001).

Two major classes of small RNAs have been characterized: shortinterfering RNAs (siRNAs) and microRNAs (miRNAs). The primarytranscripts that eventually form miRNAs are transcribed fromnon-protein-coding miRNA genes. These transcripts form hairpinstructures that are then processed by Dicer (or by Dicer-like activitiesin plants) to yield small RNA duplexes containing 2-base overhangs ateach 3′ end. The mature single-stranded miRNA approximately 20-22nucleotides in length forms by dissociation of the two strands in theduplex, and is selectively incorporated into the RNA-Induced SilencingComplex, or RISC (Zamore, Science, 296:1265-1269, 2002; Tang et al.,Genes Dev., 17:49-63, 2003; Xie et al., Curr. Biol. 13:784-789, 2003).

siRNAs are similar in chemical structure to miRNAs, however siRNAs aregenerated by the cleavage of relatively long double-stranded RNAmolecules by Dicer or DCL enzymes (Zamore, Science, 296:1265-1269, 2002;Bernstein et al., Nature, 409:363-366, 2001). In animals and plants,siRNAs are assembled into RISC and guide the sequence specificribonucleolytic activity of RISC, thereby resulting in the cleavage ofmRNAs, viral RNAs or other RNA target molecules in the cytoplasm. In thenucleus, siRNAs also guide heterochromatin-associated histone and DNAmethylation, resulting in transcriptional silencing of individual genesor large chromatin domains.

MicroRNAs in plants and animals function as posttranscriptionalregulators of genes involved in a wide range of cellular processes(Bartel, Cell 116:281-297, 2004; He & Hannon, Nat Rev Genet. 5:522-531,2004). In the plant Arabidopsis thaliana, miRNAs regulate mRNAs encodingat least twelve families of transcription factors, several miRNAmetabolic factors, and proteins involved in stress responses,metabolism, and hormone signaling (Jones-Rhoades & Bartel, Mol Cell14:787-799, 2004; Kasschau et al., Dev Cell 4:205-217, 2003; Llave etal., Science 297:2053-2056, 2002b; Vazquez et al., Curr Biol 14:346-351,2004a; Xie et al., Curr Biol 13:784-789, 2003). Plant miRNAs target adisproportionately high number of genes with functions in developmentalprocesses, including developmental timing, control of cellproliferation, meristem cell function, and patterning. Global disruptionof miRNA biogenesis or function, or specific disruption of miRNA-targetinteractions, can result in severe developmental abnormalities (Achardet al., Development 131:3357-3365, 2004; Chen, Science 303:2022-2025,2004; Emery et al., Curr Biol 13:1768-1774, 2003; Juarez et al., Nature428:84-88, 2004; Kidner & Martienssen, Nature 428:81-84, 2004; Laufs etal., Development 131:4311-4322, 2004; Mallory et al., Curr Biol14:1035-1046, 2004; Palatnik et al., Nature 425:257-263, 2003; Tang etal., Genes & Dev 17:49-63 2003; Vaucheret et al., Genes Dev18:1187-1197, 2004), indicating that miRNA-based regulation is crucialfor normal growth and development. This idea is reinforced by theconservation of most miRNAs and their corresponding targets throughsignificant evolutionary time (Bartel, Cell 116:281-297, 2004).MicroRNAs have been identified by direct cloning methods andcomputational prediction strategies (Jones-Rhoades & Bartel, Mol Cell14:787-799, 2004; Llave et al., Plant Cell 14:1605-1619, 2000a; Park etal., Curr Biol 12:1484-1495, 2002; Reinhart et al., Genes Dev16:1616-1626, 2002; Sunkar & Zhu, Plant Cell 16:2001-2019, 2004).

Plant miRNAs usually contain near-perfect complementarity with targetsites, which are found most commonly in protein-coding regions of thegenome. As a result, most (but not all) plant miRNAs function to guidecleavage of targets through a mechanism similar to the siRNA-guidedmechanism associated with RNAi (Jones-Rhoades & Bartel, Mol Cell14:787-799, 2004; Kasschau et al., Dev Cell 4:205-217, 2003; Llave etal., Science 297:2053-2056, 2002; Tang et al., Genes & Dev 17:49-632003). In contrast, animal miRNAs contain relatively low levels ofcomplementarity to their target sites, which are most commonly found inmultiple copies within 3′ untranslated regions of the target transcript(Lewis et al., Cell 115:787-798, 2003; Rajewsky & Socci, Dev Biol267:529-535, 2004; Stark et al., PLoS Biol 1:E60, 2003). Most animalmiRNAs do not guide cleavage, but rather function to repress expressionat the translational or co-translational level (Ambros, Cell113:673-676, 2003; He & Hannon, Nat Rev Genet. 5:522-531, 2004). Atleast some plant miRNAs may also function as translational repressors(Aukerman & Sakai, Plant Cell 15:2730-2741, 2003; Chen, Science303:2022-2025, 2004). Translation repression is not an inherent activityof animal miRNAs, as miRNAs will guide cleavage if presented with atarget containing high levels of complementarity (Doench et al., GenesDev 17:438-442, 2003; Hutvagner & Zamore, Science 297:2056-2060, 2002;Yekta et al., Science 304:594-596, 2004; Zeng et al., Proc Natl Acad SciUSA 100:9779-9784, 2003).

MicroRNAs form through nucleolytic maturation of genetically defined RNAprecursors that adopt imperfect, self-complementary foldback structures.Processing yields a duplex intermediate (miRNA/miRNA*) that ultimatelyprovides the miRNA strand to the effector complex, termed RISC (Khvorovaet al., Cell 115:209-216, 2003; Schwarz et al., Cell 115:199-208, 2003).Plants contain four DICER-LIKE (DCL) proteins, one of which (DCL1) isnecessary for maturation of most or all miRNA precursors (Kurihara &Watanabe, Proc Natl Acad Sci USA 101:12753-12758, 2004; Park et al.,Curr Biol 12:1484-1495, 2002; Reinhart et al., Genes Dev 16:1616-1626,2002; Schauer et al., Trends Plant Sci 7:487-491, 2002). The DCL1protein contains an RNA helicase and two RNaseIII-like domains, acentral PAZ domain and C-terminal dsRNA binding motifs. Animal miRNAprecursor processing requires Drosha, another RNaseIII domain protein,and Dicer in sequential nucleolytic steps (Lee et al., Nature425:415-419, 2003). HEN1 participates in miRNA biogenesis or stabilityin plants via a 3′ methylase activity (Boutet et al., Curr Biol13:843-848, 2003; Park et al., Curr Biol 12:1484-1495, 2002). ThedsRNA-binding HYL1 protein is necessary for miRNA biogenesis incooperation with DCL1 and HEN1 in the nucleus. Based on sequencesimilarity, HYL1 has been suggested to function like animal R2D2, whichis required post-processing during RISC assembly (Han et al., Proc NatlAcad Sci USA 101:1093-1098, 2004; Liu et al., Science 301:1921-1925,2003; Pham et al., Cell 117:83-94, 2004; Tomari et al., Science306:1377-1380, 2004; Vazquez et al., Curr Biol 14:346-351, 2004a). Inanimals, Exportin-5 (Exp5) regulates the transport of pre-miRNAs fromthe nucleus to the cytoplasm by a Ran-GTP-dependent mechanism (Bohnsacket al., RNA 10:185-191, 2004; Lund et al., Science 303:95-98, 2003; Yiet al., Genes Dev 17:3011-3016, 2003). In Arabidopsis, HST may provide arelated function to transport miRNA intermediates to the cytoplasm(Bollman et al., Development 130:1493-1504, 2003). ActivemiRNA-containing RISC complexes in plants almost certainly contain oneor more ARGONAUTE proteins, such as AGO1 (Fagard et al., Proc Natl AcadSci USA 97:11650-11654, 2000; Vaucheret et al., Genes Dev 18:1187-1197,2004). Argonaute proteins in animals were shown recently to provide thecatalytic activity for target cleavage (Liu et al., Science305:1437-1441, 2004; Meister et al., Mol Cell 15:185-197, 2004).

In addition to miRNAs, plants also produce diverse sets of endogenous21-25 nucleotide small RNAs. Most of these differ from miRNAs in thatthey arise from double-stranded RNA (rather than imperfect foldbackstructures), in some cases generated by the activity of RNA-DEPENDENTRNA POLYMERASEs (RDRs). Arabidopsis DCL2, DCL3, DCL4, RDR1, RDR2 andRDR6 have known roles in siRNA biogenesis (Dalmay et al., Cell101:543-553, 2000; Mourrain et al., Cell 101:533-542, 2000; Peragine etal., Genes & Dev 18:2369-2379, 2004; Vazquez et al., Mol Cell 16:69-79,2004b; Xie et al., PLoS Biol 2:642-652, 2004; Yu et al., Mol PlantMicrobe Interact 16:206-216, 2003). For example, DCL3 and RDR2 cooperatein the heterochromatin-associated RNAi pathway, resulting in˜24-nucleotide siRNAs from various retroelements and transposons, 5SrDNA loci, endogenous direct and inverted repeats, and transgenescontaining direct repeats (Xie et al., PLoS Biol 2:642-652, 2004;Zilberman et al., Science 299:716-719, 2003). RDR6 functions inposttranscriptional RNAi of sense transgenes, some viruses, and specificendogenous mRNAs that are targeted by trans-acting siRNAs (ta-siRNAs)(Dalmay et al., Cell 101:543-553, 2000; Mourrain et al., Cell101:533-542, 2000; Peragine et al., Genes & Dev 18:2369-2379, 2004;Vazquez et al., Mol Cell 16:69-79, 2004b; Yu et al., Mol Plant MicrobeInteract 16:206-216, 2003). Ta-siRNAs arise from transcripts that arerecognized by RDR6, in cooperation with SGS3, as a substrate to formdsRNA. The dsRNA is processed accurately in 21-nucleotide steps by DCL1to yield a set of “phased” ta-siRNAs. These ta-siRNAs interact withtarget mRNAs to guide cleavage by the same mechanism as do plant miRNAs(Peragine et al., Genes & Dev 18:2369-2379, 2004; Vazquez et al., MolCell 16:69-79, 2004).

There is a need to develop methods and constructs that can be used toinduce targeted RNAi in vivo. It is to such methods and constructs, andrelated compositions, that this disclosure is drawn.

SUMMARY OF THE DISCLOSURE

Provided herein are methods of generating one or more siRNAs in vivo;also provided are constructs and compositions useful in the methods. Themethods do not depend on DNA or other synthetic nucleic acid moleculesthat contain inverted duplications (repeats) or dual promoters to formperfect or largely double-stranded RNA. Rather, the methods employconstructs that yield single-stranded RNA transcripts, and takeadvantage of endogenous (native or heterologous) or in vivo-producedmiRNAs or siRNAs to initiate production of siRNAs from an engineeredRNAi-triggering cassette. The miRNAs or siRNAs guide cleavage of thetranscript and set the register (phase) for production of siRNAs(usually 21 nucleotides in length) encoded adjacent to the initiationcleavage site within the construct. The methods result in specificformation of siRNAs of predictable size and register (phase) relative tothe initiation cleavage site. The method can be used to produce specificsiRNAs in vivo for inactivation or suppression of one or more targetgenes or other entities, such as pathogens or pests (e.g., viruses,bacteria, nematodes). No exogenous hairpin or foldback structure isrequired in the provided constructs in order to generate siRNAs or tocarry out RNAi-like inhibition of target gene(s).

Also provided are methods, and constructs for use in such methods, wherethe siRNAs are produced in a tissue-specific, cell-specific, or otherregulated manner.

Further, transformed cells and organisms that contain a transgeneincluding at least one RNAi-triggering cassette are also provided bythis disclosure. For instance, transgenic fungi, invertebrate animals,and plants are provided that contain at least one RNAi-triggeringcassette, which, when transcribed, produces at least one siRNA moleculecomplementary to a target sequence to be inhibited in that organism.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-1D. Refined prediction and validation of miRNA target genes inArabidopsis. (FIG. 1A) Flowchart for miRNA target identification. Thenumber of small RNAs (or targets) passing a filter is shown inparentheses. Predicted targets are classified into 5 bins based onvalidation data. The false negative rate in Bins 1 and 2 are based on 66and 28 targets in the ‘Rule development set’, respectively (see Table3). (FIG. 1B) Percent of mismatched and G:U base-pairs at each positionof the Rule development set targets. Position 1 corresponds to the 5′end of the miRNA. (FIG. 1C) Minimum Free Energy (MFE) ratio of the Ruledevelopment set target duplexes. Black circles indicate Rule developmentset validated targets, open circles indicate rule development settargets only predicted computationally. (FIG. 1D) Number of predictedtargets genes for a given miRNA-target duplex score, filtered byduplexes with an MFE ratio ≧0.73. Total predicted targets (opentriangles) and captured targets in the Rule development set (opencircles) are shown. Total targets in the Rule development set (94) isindicated by the dashed line.

FIG. 2A-2B. miRNA-target duplexes. (FIG. 2A) Target duplexes from Bin 1validated in this study. (FIG. 2B) Duplexes for predicted miRNA targetsin Bin 3.

FIG. 3A-3B. Validation of miRNA targets by 5′RACE. (FIG. 3A)Protein-coding miRNA targets. The miRNA-target duplex is highlighted,with the fraction of cloned PCR products terminating at a given positionin the target validation assay (Llave et al., Science 297:2053-2056,2002) indicated above the duplex. The distribution of cleavage productsacross all five predicted miR399 target sites is displayed above theschematic representation of At2g33770. (FIG. 3B) Non-coding miRNAtargets predicted by the EST database search. Each of these targetscorresponds to a ta-siRNA-generating primary transcript.

FIG. 4A-G. Characterization and expression profiling of Arabidopsissmall RNA biogenesis mutants. (FIG. 4A) Phenotype of hst-15 and rdr6-15mutants. Rosettes (Col-0, rdr6-15, hst-15), first true leaf (Col-0,rdr6-15), bolt and flower (Col-0, hst-15) are shown. For array data in(b-e), normalized intensity is plotted as log₂ of the fold changerelative to the control sample for each mutant, thus zero represents nochange in transcript abundance. (FIG. 4B) Profile of 81 of 94 miRNAtarget transcripts predicted previously and in this study (Bins 1 and 2,FIG. 1). (FIG. 4C) Profile of 12 of 18 miRNA targets genes predicted inthis study. The solid lines indicate new targets from existing targetfamilies (Bin 3, FIG. 1), and the dashed lines indicate novel miRNAtargets (Bin 4, FIG. 1). Non-validated targets in Bin 5 are not shown.(FIG. 4D) Profile of transcripts significantly co-affected (P>0.01) indcl1-7, hen1-1, and rdr6-15. (FIG. 4E) Profile of 93 predicted miRNAtarget transcripts (light lines), and PCA component 1 (dark line). (FIG.4F) Cladogram of the small RNA biogenesis mutant series. The correlationamong groups (r×100) is shown at each node. (FIG. 4G) Scatterplots ofall genes showing normalized intensity values representing fold change(hyl1-2 vs. hst-15, dcl1-7 vs. hen1-1, hyl1-2 vs. dcl3-1).

FIG. 5A-E. In-phase processing of trans-acting siRNAs directed by miR173as the initiator. (FIG. 5A-5C) Diagrammatic representation of the threeTAS1 and on TAS2 loci. The naming convention used is TAS (forTrans-Acting SiRNA). Ta-siRNAs with functional evidence are shown by thesystematic nomenclature (see text for details). The 21 nucleotide phaseis indicated by brackets, with the first position starting from themiR173-guided cleavage site. The relative positions from the cleavagesite are designated 3′D1, 3′D2, etc. Positions for which small RNAs arerepresented in the ASRP sequence database are listed with the ASRP IDnumber. Relative positions of the At2g39675 and At3g39680 loci inArabidopsis chromosome 2 are shown in (FIG. 5C). (FIG. 5D) Detection andvalidation of ta-siRNAs by small RNA blot analysis. Small RNAs weredetected using specific oligo probes, except At3g39680 antisense smallRNAs which were detected with a 469 nucleotide radiolabeled RNAtranscript. (FIG. 5E) Validation of siR255 target genes by 5′ RACE, andpredicted Ag3g39681 (TAS2).3′D6(−) targeted PPR genes.

FIG. 6A-E. In-phase processing of TAS3-derived trans-acting siRNAsguided by miR390. (FIG. 6A) Diagrammatic representation of the miR390target locus, TAS3 (At3g17185). Labeling is as in FIG. 5, but with the21-nucleotide phased positions designated 5′D1, 5′D2, etc., starting atthe miR390-guided cleavage site. The two siRNAs that are predicted toguide cleavage of ARF3 and ARF4 are indicated. (FIG. 6B) Detection andvalidation of ta-siRNAs from the TAS3 locus. (FIG. 6C) T-Coffee programalignment of TAS3 orthologs in plants showing conservation of predictedTAS3 ta-siRNAs and miR390 target site. High levels of conservation aredesignated by light shading. (FIG. 6D) PLOTCON program similarity score(21 nt window) derived from alignment of 18 ARF3 and ARF4 genes from 16species, over a 600 nt region. Two highly conserved regions areindicated by A and B, which are TAS3 ta-siRNA target sites. Below,validation of small RNA directed cleavage of ARF3 and ARF4 by 5′ RACE.The predicted TAS3-derived ta-siRNAs are shown below complementaryregions of ARF3 and ARF4 sequences. (FIG. 6E) Consensus phylogenetictree of the ARF family, showing miRNA and ta-siRNA regulated branches.Bayesian posterior probability was 100 except for labeled nodes.

FIG. 7. Model for miRNA-directed formation of ta-siRNAs

FIG. 8A-D. Validation of miRNAs in A. thaliana. (FIG. 8A) Predictionflowchart for miRNA validation. The number of small RNAs passing afilter is shown in parentheses. (FIG. 8B) Predicted secondary structureof miRNA precursors validated in this study. (FIG. 8C, FIG. 8D) SmallRNA blot analysis of miRNAs. miR159 and miR167 are shown as traditionalmiRNA controls, AtSN1 is shown as an siRNA control. Ethidiumbromide-stained gel (tRNA and 5S RNA zone) is shown at the bottom.Wildtype controls (Col-0 and La-er) are shown next to respective miRNAmetabolism mutants (FIG. 8C) and ta-siRNA biogenesis mutants (rdr6-11and sgs3-11) or transgenic plants expressing viral silencing (FIG. 8D).

FIG. 9A-B. Strategy to map Arabidopsis MIRNA gene transcription startsites. (FIG. 9A) Schematic representation of a generic MIRNA transcript(top), and control SCL6-IV mRNA (middle) and miR171-guided cleavageproduct (bottom). The relative positions of oligonucleotides used in5′RACE reactions are shown. (FIG. 9B) RLM-5′RACE reactions usingpoly(A)⁺-selected RNA that was pretreated with calf intestinalphosphatase (CIP) plus tobacco acid pyrophosphatase (TAP, even-numberedlanes) or with buffer (odd-numbered lanes) prior to adaptor ligation.The 5′RACE products for SCL6-IV-specific RNAs (lanes1-4) and three MIRNAloci (lanes 5-10) were resolved on a 2% agarose gel. Gene-specificprimers used for 5′RACE are indicated above each lane.

FIG. 10A-C. MIRNA gene transcript start sites and core promoterelements. (FIG. 10A) Base frequency at MIRNA transcription initiationsites (n=63). (FIG. 10B) Genomic sequences (−50 to +10 relative to startsites) around 63 start sites (red letters) from 47 Arabidopsis MIRNAloci. Putative TATA motifs (bold) are indicated. These sequencescorrespond to SEQ ID NOs: 286-348. (FIG. 10C) Occurrence of high-scoringTATA motifs within a 250-nucleotide (−200 to +50) genomic context for 63MIRNA transcripts.

FIG. 11. Graphic representation of an artificial ta-siRNA construct madein the TAS1c context. The construct contains two 21-nt siRNA modules.The represented construct contains siRNAs designed to target mRNAs forArabidopsis phytoene desaturase (PDS).

FIG. 12A-D. Demonstration of artificial ta-siRNA biogenesis and activityin Nicotiana benthamiana. Introduction of each construct into N.benthamiana in a transient assay resulted in miR173-dependent formationof ta-siRNAs. In the case of 35S:TAS1cGFPd3d4 (FIG. 12A,B), theartificial ta-siRNA construct was co-expressed with a functional GFPgene. Expression of at least one artificial tasiRNA was detected in amiR173-dependent manner, by blot assay using each construct (GFP: FIG.12A; PDS: FIG. 12C; PID: FIG. 12D). The GFP gene was silenced by theartificial GFP ta-siRNAs in a miR173-dependent manner (FIG. 12B). Thesame miR173 and tasiRNA255 controls were used for PDS, PID, and GFPsiRNA assays)

FIG. 13. Artificial ta-siRNA biogenesis and activity in transgenicArabidopsis. The PDS artificial ta-siRNA-generating construct wasintroduced into wild-type (Col-0) Arabidopsis and rdr6-15 mutant plants.Both strong and weak loss-of-function PDS phenotypes were detected, butonly in wt plants. The rdr6-15 mutant plants lack a critical factor forta-siRNA biogenesis.

FIG. 14A-C. Reconstruction of TAS1a, TAS1b, TAS1c, and TAS2 ta-siRNABiogenesis in a Transient Expression Assay using N. benthamiana. (FIG.14A and FIG. 14B) Constructs with wild-type miR173 target sites.Constructs were expressed or coexpressed as indicated above the blotpanels. The small RNAs detected in blot assays are shown to the right ofeach panel. Duplicate biological samples were analyzed for mosttreatments. (FIG. 14C) Constructs with mutagenized target site or miR173sequences. Target site and miRNA combinations tested are illustratedschematically above the blot panels. Mutagenized positions are in bold.The miR173res1 probe hybridized to both the miR173 and miR173res1sequences.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

The nucleic acid sequences listed below are shown using standard letterabbreviations for nucleotide bases, as defined in 37 C.F.R. §1.822. Onlyone strand of each nucleic acid sequence is shown, but the complementarystrand is understood as included in embodiments where it would beappropriate. The Sequence Listing is submitted as an ASCII text filenamed SeqList.txt, created on Apr. 24, 2014, ˜120 KB, which isincorporated by reference herein. In the accompanying Sequence Listing:

SEQ ID NOs: 1-142 and 281-285 are representative target (initiator)sequences. The initiator sequences are shown as RNA; it is understoodthat the corresponding DNA sequence would comprise a T in place of anyU. The sequences are broken out based on the miRNA complementary to theprovided initiator (target) sequences. The corresponding miRNA sequencecan be deduced for each target sequence; it is the reverse complementformed of RNA.

SEQ ID NOs: 143-154 are predicted miRNA candidates (shown as RNA) thatwere tested experimentally, and which are discussed in Example 5.

SEQ ID NOs: 155-206 are miRNA sequences (shown as RNA), which arediscussed in Example 5.

SEQ ID NOs: 207-276 are validated miRNA sequences cloned fromArabidopsis small RNA libraries (shown as RNA), and which are discussedin Example 5.

SEQ ID NO: 277 is the nucleic acid sequence of an artificial ta-siRNAlocus targeting Arabidopsis gene encoding GFP.

SEQ ID NO: 278 is the nucleic acid sequence of an artificial ta-siRNAlocus targeting Arabidopsis gene encoding phytoene desaturase (PDS).

SEQ ID NO: 279 is the nucleic acid sequence of an artificial ta-siRNAlocus targeting Arabidopsis gene encoding PINOID (PID).

SEQ ID NO: 280 is an example of a sequence that would be contained inDNA construct containing SEQ ID NO: 1 as an initiator sequence.

SEQ ID NOs: 286-348 are genomic sequences (−50 to +10 relative to startsites) of 63 start sites in 47 Arabidopsis miRNA loci. These are showngraphically in FIG. 10B.

SEQ ID NOs: 349 to 614 are primers used in 3′RACE confirmationsequencing.

DETAILED DESCRIPTION I. Abbreviations

AGO Argonaute

asRNA antisense RNA

cDNA complementary DNA

DCL dicer-like

dsRNA double-stranded RNA

GFP green fluorescent protein

LKR lysine ketoglutarate reductase

miRNA microRNA

nt nucleotide

PID PINOID

PDS phytoene desaturase

PTGS post-transcriptional gene silencing

RDR RNA-dependent RNA polymerase

RISC RNA-induced silencing complex

RNAi RNA interference

siRNA small interfering RNA

ssRNA single stranded RNA

ta-siRNA trans-acting siRNA

TGS transcriptional gene silencing

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, by VCH Publishers,Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of theinvention, the following non-limiting explanations of specific terms areprovided:

21-Nucleotide Phasing: An incremental 21-nucleotide register, startingat an initiator cleavage site, in which cleavage is mediated by a RISCguided by either a miRNA or siRNA. Phasing defines a set of 21nucleotide segments in linear, end-to-end orientation, either to the 5′or 3′ side of the initiator cleavage site, or both. Formation of the21-nucleotide siRNAs in phase with the cleavage site depends on theactivity of a DICER or DICER-LIKE enzyme.

Agent: Any substance, including, but not limited to, an antibody,chemical compound, small molecule, therapeutic, nucleic acid, peptidemimetic, peptide, or protein. An agent can increase or decrease thelevel of miRNA or siRNA expression or production.

Agronomic trait: Characteristic of a plant, which characteristicsinclude, but are not limited to, plant morphology, physiology, growthand development, yield, nutritional enhancement, disease or pestresistance, or environmental or chemical tolerance are agronomic traits.In the plants of this disclosure, the expression of identifiedrecombinant DNA, e.g. for gene suppression, confers an agronomicallyimportant trait, e.g. increased yield. An “enhanced agronomic trait”refers to a measurable improvement in an agronomic trait including, butnot limited to, yield increase, including increased yield undernon-stress conditions and increased yield under environmental stressconditions. Stress conditions may include, for example, drought, shade,fungal disease, viral disease, bacterial disease, insect infestation,nematode infestation, cold temperature exposure, heat exposure, osmoticstress, reduced nitrogen nutrient availability, reduced phosphorusnutrient availability and high plant density. “Yield” can be affected bymany properties including without limitation, plant height, pod number,pod position on the plant, number of internodes, incidence of podshatter, grain size, efficiency of nodulation and nitrogen fixation,efficiency of nutrient assimilation, resistance to biotic and abioticstress, carbon assimilation, plant architecture, resistance to lodging,percent seed germination, seedling vigor, and juvenile traits. Yield canalso affected by efficiency of germination (including germination instressed conditions), growth rate (including growth rate in stressedconditions), ear number, seed number per ear, seed size, composition ofseed (starch, oil, protein) and characteristics of seed fill. Increasedyield may result from improved utilization of key biochemical compounds,such as nitrogen, phosphorous and carbohydrate, or from improvedresponses to environmental stresses, such as cold, heat, drought, salt,and attack by pests or pathogens. Recombinant DNA used in thisdisclosure can also be used to provide plants having improved growth anddevelopment, and ultimately increased yield, as the result of modifiedexpression of plant growth regulators or modification of cell cycle orphotosynthesis pathways.

Altering level of production or expression: Changing, either byincreasing or decreasing, the level of production or expression of anucleic acid sequence or an amino acid sequence (for example apolypeptide, an siRNA, a miRNA, an mRNA, a gene), as compared to acontrol level of production or expression.

Antisense, Sense, and Antigene: DNA has two antiparallel strands, a5′→3′ strand, referred to as the plus strand, and a 3′→5′ strand,referred to as the minus strand. Because RNA polymerase adds nucleicacids in a 5′→3′ direction, the minus strand of the DNA serves as thetemplate for the RNA during transcription. Thus, an RNA transcript willhave a sequence complementary to the minus strand, and identical to theplus strand (except that U is substituted for T).

Antisense molecules are molecules that are specifically hybridizable orspecifically complementary to either RNA or the plus strand of DNA.Sense molecules are molecules that are specifically hybridizable orspecifically complementary to the minus strand of DNA. Antigenemolecules are either antisense or sense molecules directed to a DNAtarget. An antisense RNA (asRNA) is a molecule of RNA complementary to asense (encoding) nucleic acid molecule.

Amplification: When used in reference to a nucleic acid, this refers totechniques that increase the number of copies of a nucleic acid moleculein a sample or specimen. An example of amplification is the polymerasechain reaction, in which a biological sample collected from a subject iscontacted with a pair of oligonucleotide primers, under conditions thatallow for the hybridization of the primers to nucleic acid template inthe sample. The primers are extended under suitable conditions,dissociated from the template, and then re-annealed, extended, anddissociated to amplify the number of copies of the nucleic acid. Theproduct of in vitro amplification can be characterized byelectrophoresis, restriction endonuclease cleavage patterns,oligonucleotide hybridization or ligation, and/or nucleic acidsequencing, using standard techniques. Other examples of in vitroamplification techniques include strand displacement amplification (seeU.S. Pat. No. 5,744,311); transcription-free isothermal amplification(see U.S. Pat. No. 6,033,881); repair chain reaction amplification (seeWO 90/01069); ligase chain reaction amplification (see EP-A-320 308);gap filling ligase chain reaction amplification (see U.S. Pat. No.5,427,930); coupled ligase detection and PCR (see U.S. Pat. No.6,027,889); and NASBA™ RNA transcription-free amplification (see U.S.Pat. No. 6,025,134).

Binding or stable binding: An oligonucleotide binds or stably binds to atarget nucleic acid if a sufficient amount of the oligonucleotide formsbase pairs or is hybridized to its target nucleic acid, to permitdetection of that binding. Binding can be detected by either physical orfunctional properties of the target:oligonucleotide complex. Bindingbetween a target and an oligonucleotide can be detected by any procedureknown to one skilled in the art, including both functional and physicalbinding assays. For instance, binding can be detected functionally bydetermining whether binding has an observable effect upon a biosyntheticprocess such as expression of a gene, DNA replication, transcription,translation and the like.

Physical methods of detecting the binding of complementary strands ofDNA or RNA are well known in the art, and include such methods as DNaseI or chemical footprinting, gel shift and affinity cleavage assays,Northern blotting, dot blotting and light absorption detectionprocedures. For example, one method that is widely used, because it issimple and reliable, involves observing a change in light absorption ofa solution containing an oligonucleotide (or an analog) and a targetnucleic acid at 220 to 300 nm as the temperature is slowly increased. Ifthe oligonucleotide or analog has bound to its target, there is a suddenincrease in absorption at a characteristic temperature as theoligonucleotide (or analog) and the target disassociate from each other,or melt.

The binding between an oligomer and its target nucleic acid isfrequently characterized by the temperature (T_(m)) at which 50% of theoligomer is melted from its target. A higher (T_(m)) means a stronger ormore stable complex relative to a complex with a lower (T_(m)).

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and transcriptional regulatory sequences. cDNA mayalso contain untranslated regions (UTRs) that are responsible fortranslational control in the corresponding RNA molecule. cDNA is usuallysynthesized in the laboratory by reverse transcription from messengerRNA extracted from cells or other samples.

Complementarity and percentage complementarity: Molecules withcomplementary nucleic acids form a stable duplex or triplex when thestrands bind, or hybridize, to each other by forming Watson-Crick,Hoogsteen or reverse Hoogsteen base pairs. Stable binding occurs when anoligonucleotide remains detectably bound to a target nucleic acidsequence under the required conditions.

Complementarity is the degree to which bases in one nucleic acid strandbase pair with (are complementary to) the bases in a second nucleic acidstrand. Complementarity is conveniently described by the percentage,i.e., the proportion of nucleotides that form base pairs between twostrands or within a specific region or domain of two strands. Forexample, if 10 nucleotides of a 15-nucleotide oligonucleotide form basepairs with a targeted region of a DNA molecule, that oligonucleotide issaid to have 66.67% complementarity to the region of DNA targeted.

Sufficient complementarity means that a sufficient number of base pairsexist between the oligonucleotide and the target sequence to achievedetectable binding, and disrupt or reduce expression of the geneproduct(s) encoded by that target sequence. When expressed or measuredby percentage of base pairs formed, the percentage complementarity thatfulfills this goal can range from as little as about 50% complementarityto full, (100%) complementary. In some embodiments, sufficientcomplementarity is at least about 50%, about 75% complementarity, or atleast about 90% or 95% complementarity. In particular embodiments,sufficient complementarity is 98% or 100% complementarity.

A thorough treatment of the qualitative and quantitative considerationsinvolved in establishing binding conditions that allow one skilled inthe art to design appropriate oligonucleotides for use under the desiredconditions is provided by Beltz et al., Methods Enzymol 100:266-285,1983, and by Sambrook et al. (ed.), Molecular Cloning: A LaboratoryManual, 2^(nd) ed., v:1-3, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

Complementary: The base pairing that occurs between two distinct nucleicacid sequences or two distinct regions of the same nucleic acidsequence.

Control level: The level of a molecule, such as a polypeptide or nucleicacid, normally found in nature under a certain condition and/or in aspecific genetic background. In certain embodiments, a control level ofa molecule can be measured in a cell or specimen that has not beensubjected, either directly or indirectly, to a treatment. A controllevel is also referred to as a wildtype or a basal level. These termsare understood by those of ordinary skill in the art.

Control plant: A control plant, i.e. a plant that does not contain arecombinant DNA that confers (for instance) an enhanced agronomic traitin a transgenic plant, is used as a baseline for comparison to identifyan enhanced agronomic trait in the transgenic plant. A suitable controlplant may be a non-transgenic plant of the parental line used togenerate a transgenic plant. A control plant may in some cases be atransgenic plant line that comprises an empty vector or marker gene, butdoes not contain the recombinant DNA, or does not contain all of therecombinant DNAs in the test plant.

DICER-LIKE (DCL): Plant homologs of the animal protein DICER. Both DICERand DCL enzymes catalyze formation of small RNA duplexes from largerprecursor RNA molecules. By way of example, Arabidopsis thalianacontains four DCL genes (DCL1-DCL4). DCL1 for instance catalyzesprocessing of fold-back precursors for miRNAs (GenBank Accession No.NM_(—)099986; locus position At1g01040).

DNA (deoxyribonucleic acid): DNA is a long chain polymer which comprisesthe genetic material of most living organisms (some viruses have genescomprising ribonucleic acid (RNA)). The repeating units in DNA polymersare four different nucleotides, each of which comprises one of the fourbases, adenine, guanine, cytosine and thymine bound to a deoxyribosesugar to which a phosphate group is attached. Triplets of nucleotides(referred to as codons) code for each amino acid in a polypeptide, orfor a stop signal. The term codon is also used for the corresponding(and complementary) sequences of three nucleotides in the mRNA intowhich the DNA sequence is transcribed.

Unless otherwise specified, any reference to a DNA molecule is intendedto include the reverse complement of that DNA molecule. Except wheresingle-strandedness is required by the text herein, DNA molecules,though written to depict only a single strand, encompass both strands ofa double-stranded DNA molecule.

Encode: A polynucleotide is said to encode a polypeptide if, in itsnative state or when manipulated by methods well known to those skilledin the art, it can be transcribed and/or translated to produce the mRNAfor and/or the polypeptide or a fragment thereof. The anti-sense strandis the complement of such a nucleic acid, and the encoding sequence canbe deduced therefrom.

Expression: The process by which a gene's coded information is convertedinto the structures present and operating in the cell. Expressed genesinclude those that are transcribed into mRNA and then translated intoprotein and those that are transcribed into RNA but not translated intoprotein (for example, siRNA, transfer RNA and ribosomal RNA). Thus,expression of a target sequence, such as a gene or a promoter region ofa gene, can result in the expression of an mRNA, a protein, or both. Theexpression of the target sequence can be inhibited or enhanced(decreased or increased).

Fluorophore: A chemical compound, which when excited by exposure to aparticular wavelength of light, emits light (i.e., fluoresces), forexample at a different wavelength than that to which it was exposed.Fluorophores can be described in terms of their emission profile, or“color.” Green fluorophores, for example Cy3, FITC, and Oregon Green,are characterized by their emission at wavelengths generally in therange of 515-540λ. Red fluorophores, for example Texas Red, Cy5 andtetramethylrhodamine, are characterized by their emission at wavelengthsgenerally in the range of 590-690λ.

Encompassed by the term “fluorophore” are luminescent molecules, whichare chemical compounds which do not require exposure to a particularwavelength of light to fluoresce; luminescent compounds naturallyfluoresce. Therefore, the use of luminescent signals eliminates the needfor an external source of electromagnetic radiation, such as a laser. Anexample of a luminescent molecule includes, but is not limited to,aequorin (Tsien, Ann. Rev. Biochem. 67:509, 1998).

Examples of fluorophores are provided in U.S. Pat. No. 5,866,366. Theseinclude: 4-acetamido-4′-isothiocyanatostilbene-2,2′ disulfonic acid,acridine and derivatives such as acridine and acridine isothiocyanate,5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS),4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5disulfonate (LuciferYellow VS), N-(4-anilino-1-naphthyl)maleimide, anthranilamide, BrilliantYellow, coumarin and derivatives such as coumarin,7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanosine;4′,6-diaminidino-2-phenylindole (DAPI);5′,5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride);4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL);4-dimethylaminophenyl-azophenyl-4′-isothiocyanate (DABITC); eosin andderivatives such as eosin and eosin isothiocyanate; erythrosin andderivatives such as erythrosin B and erythrosin isothiocyanate;ethidium; fluorescein and derivatives such as 5-carboxyfluorescein(FAM), dichlorotriazin-2-yl)aminofluorescein (DTAF),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate (FITC), and QFITC (XRITC); fluorescamine;IR144; IR1446; Malachite Green isothiocyanate; 4-methylumbelliferone;ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such aspyrene, pyrene butyrate and succinimidyl 1-pyrene butyrate; Reactive Red4 (Cibacron® Brilliant Red 3B-A); rhodamine and derivatives such as6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acidand terbium chelate derivatives.

Other fluorophores include thiol-reactive europium chelates that emit atapproximately 617 nm (Heyduk and Heyduk, Analyt. Biochem. 248:216-227,1997; J. Biol. Chem. 274:3315-3322, 1999).

Still other fluorophores include cyanine, merocyanine, styryl, andoxonyl compounds, such as those disclosed in U.S. Pat. Nos. 5,268,486;5,486,616; 5,627,027; 5,569,587; and 5,569,766, and in published PCTpatent application no. US98/00475, each of which is incorporated hereinby reference. Specific examples of fluorophores disclosed in one or moreof these patent documents include Cy3 and Cy5, for instance.

Other fluorophores include GFP, Lissamine™, diethylaminocoumarin,fluorescein chlorotriazinyl, naphthofluorescein, 4,7-dichlororhodamineand xanthene (as described in U.S. Pat. No. 5,800,996 to Lee et al.,herein incorporated by reference) and derivatives thereof. Otherfluorophores are known to those skilled in the art, for example thoseavailable from Molecular Probes (Eugene, Oreg.).

Gene Silencing: Gene silencing refers to lack of (or reduction of) geneexpression as a result of, though not limited to, effects at a genomic(DNA) level such as chromatin re-structuring, or at thepost-transcriptional level through effects on transcript stability ortranslation. Current evidence suggests that RNA interference (RNAi) is amajor process involved in transcriptional and posttranscriptional genesilencing.

Because RNAi exerts its effects at the transcriptional and/orpost-transcriptional level, it is believed that RNAi can be used tospecifically inhibit alternative transcripts from the same gene.

Heterologous: A type of sequence that is not normally (i.e. in thewild-type sequence) found adjacent to a second sequence. In oneembodiment, the sequence is from a different genetic source, such as avirus or organism, than the second sequence.

Hybridization: Oligonucleotides and their analogs hybridize by hydrogenbonding, which includes Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary bases. Generally, nucleic acidconsists of nitrogenous bases that are either pyrimidines (cytosine (C),uracil (U), and thymine (T)) or purines (adenine (A) and guanine (G)).These nitrogenous bases form hydrogen bonds between a pyrimidine and apurine, and the bonding of the pyrimidine to the purine is referred toas base pairing. More specifically, A will hydrogen bond to T or U, andG will bond to C. In RNA molecules, G also will bond to U. Complementaryrefers to the base pairing that occurs between two distinct nucleic acidsequences or two distinct regions of the same nucleic acid sequence.

Hybridization conditions resulting in particular degrees of stringencywill vary depending upon the nature of the hybridization method ofchoice and the composition and length of the hybridizing nucleic acidsequences. Generally, the temperature of hybridization and the ionicstrength (especially the Na⁺ concentration) of the hybridization bufferwill determine the stringency of hybridization, though waste times alsoinfluence stringency. Calculations regarding hybridization conditionsrequired for attaining particular degrees of stringency are discussed bySambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed.,vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989, chapters 9 and 11, herein incorporated by reference. The followingis an exemplary set of hybridization conditions and is not meant to belimiting.

Very High Stringency (Detects Sequences that Share 90% SequenceIdentity)

Hybridization: 5×SSC at 65° C. for 16 hours

Wash twice: 2×SSC at room temperature (RT) for 15 minutes each

Wash twice: 0.5×SSC at 65° C. for 20 minutes each

High Stringency (Detects Sequences that Share 80% Sequence Identity orGreater)

Hybridization: 5×-6×SSC at 65° C.-70° C. for 16-20 hours

Wash twice: 2×SSC at RT for 5-20 minutes each

Wash twice: 1×SSC at 55° C.-70° C. for 30 minutes each

Low Stringency (Detects Sequences that Share Greater than 50% SequenceIdentity)

Hybridization: 6×SSC at RT to 55° C. for 16-20 hours

Wash at least twice: 2×-3×SSC at RT to 55° C. for 20-30 minutes each.

Initiator sequence: A nucleotide sequence of about 21 nucleotides inlength that, when present in an RNA molecule, serves as a cleavage sitethat is recognized by a RISC guided by a miRNA or siRNA. Cleavage at aninitiator sequence (usually between the tenth and eleventh nucleotidecounted from the 3′ end of the initiator sequence) sets the21-nucleotide phasing within one or both RNA molecules that result aftercleavage. These cleavage products, after conversion to double-strandedRNA, are subject to processing by Dicer or DCL enzymes usually in21-nucleotide intervals upstream and/or downstream of the initiatorsequence. In an engineered nucleic acid cassette as described herein,such in-phase cleavages release siRNAs from the cassette. Representativeinitiator sequences, also referred to as miRNA target sequences, areshown herein, including SEQ ID NOs: 1-142 and 281-285. Additionalinitiator sequences will be known to those of ordinary skill in the art.See, for instance, sequences listed in the public databasemiRBase::Sequences (available on-line through the Sanger Institutewebsite, microma.sanger.ac.uk/sequences/index.shtml); sequences in thatdatabase through Release 7.1 (October 2005) are included herein byreference.

In the following table of target (initiator) sequences (Table 1), theinitiator cleavage site is indicated by a “˜” symbol. The sequences arebroken out based on the miRNA families. The corresponding miRNAsequence(s) or miRNA family sequences are largely complementary to thetarget sequences. The gene name indicates a representative plant speciesfor each sequence: At=Arabidopsis thaliana; Gh=Gossypium hirsutum;Gm=Glycine max; Hv=Hordeum vulgare; Le=Lycopersicum esculentum; Lj=Lotusjaponicus; Mc=Mesembryanthemum crystallinum; Mt=Medicago truncatula;Os=Oryza sativa; Pg=Pennisetum glaucum; Pt=Populus tremula; Pv=Plumbagozeylanica; Sb=Sorghum bicolor; So=Saccharum officinarum; Tc=Theobromacacao; Ta=Triticum aesitivum; Vv=Vitis vinifera; Zm=Zea mays. Additionalplants containing these sequences are discussed below. Validated miRNAtarget sequences have been confirmed experimentally using a cleavagesite assay (Llave et al., Science 297:2053-2056, 2002); predictedsequences have yet to be examined experimentally and identified in vivo,but were predicted computationally. Sequences that are known only inArabidopsis are indicated.

TABLE 1 SEQ ID Gene Target Sequence Status #miR156 family target sequences - all plants At1g27370GUGCUCUCUC~UCUUCUGUCA Validated   1 At1g53160 CUGCUCUCUC~UCUUCUGUCAValidated   2 At2g33810 UUGCUUACUC~UCUUCUGUCA Predicted   3 At3g15270CCGCUCUCUC~UCUUCUGUCA Predicted   4miR159 family target sequences - all plants At5g06100UGGAGCUCCCU~UCAUUCCAAU Validated   5 At2g26960 UCGAGUUCCCU~UCAUUCCAAUPredicted   6 At4g26930 AUGAGCUCUCU~UCAAACCAAA Predicted   7 At2g26950UGGAGCUCCCU~UCAUUCCAAG Predicted   8 At2g32460 UAGAGCUUCCU~UCAAACCAAAPredicted   9 At3g60460 UGGAGCUCCAU~UCGAUCCAAA Predicted  10 At5g55020AGCAGCUCCCU~UCAAACCAAA Predicted  11 PvMYB CAGAGCUCCCU~UCACUCCAAUPredicted  12 VvMYB UGGAGCUCCCU~UCACUCCAAU Predicted  13 HvMYB33UGGAGCUCCCU~UCACUCCAAG Predicted  14 OsMYB33 UGGAGCUCCCU~UUAAUCCAAUPredicted  15 miR160 family target sequences - all plants At1g77850UGGCAUGCAGG~GAGCCAGGCA Validated  16 At2g28350 AGGAAUACAGG~GAGCCAGGCAValidated  17 At4g30080 GGGUUUACAGG~GAGCCAGGCA Validated  18 OsARFAGGCAUACAGG~GAGCCAGGCA Predicted  19 LjARF AAGCAUACAGG~GAGCCAGGCAPredicted  20 miR161 family target sequences - Arabidopsis At5g41170ACCUGAUGUAA~UCACUUUCAA Validated  21 At1g06580 CCCGGAUGUAA~UCACUUUCAGValidated  22 At1g63150 UUGUUACUUUC~AAUGCAUUGA Validated  23 At5g16640CCCUGAUGUAU~UUACUUUCAA Predicted  24 At1g62590 UAGUCACGUUC~AAUGCAUUGAPredicted  25 At1g62670 CCCUGAUGUAU~UCACUUUCAG Predicted  26 At1g62860CCCUGAUGUUG~UUACUUUCAG Predicted  27 At1g62910 UAGUCACUUUC~AGCGCAUUGAPredicted  28 At1g62930 UCCAAAUGUAG~UCACUUUCAG Predicted  29 At1g63080UCCAAAUGUAG~UCACUUUCAA Predicted  30 At1g63130 UCCAAAUGUAG~UCACUUUCAGPredicted  31 At1g63400 UCCAAAUGUAG~UCACUUUCAA Predicted  32 At1g63230UUGUAACUUUC~AGUGCAUUGA Predicted  33 At1g63330 UAGUCACGUUC~AAUGCAUUGAPredicted  34 At1g63630 UUGUUACUUUC~AGUGCAUUGA Predicted  35 At1g64580CCCUGAUGUUG~UCACUUUCAC Predicted  36 At2g41720 UUGUUACUUAC~AAUGCAUUGAPredicted  37 At1g63070 UAGUCUUUUUC~AACGCAUUGA Predicted  38miR162 family target sequences - all pl ants At1g01040CUGGAUGCAGA~GGUAUUAUCGA Validated  39 PtDCL1 CUGGAUGCAGA~GGUCUUAUCGAPredicted  40 OsDCL1 CUGGAUGCAGA~GGUUUUAUCGA Predicted  41miR163 family target sequences - Arabidopsis At1g66700AUCGAGUUCCAAG~UCCUCUUCAA Validated  42 At1g66720AUCGAGUUCCAGG~UCCUCUUCAA Validated  43 At3g44860AUCGAGUUCCAAG~UUUUCUUCAA Validated  44miR164 family target sequences - all plants At1g56010AGCACGUACCC~UGCUUCUCCA Validated  45 At5g07680 UUUACGUGCCC~UGCUUCUCCAValidated  46 At5g53950 AGCACGUGUCC~UGUUUCUCCA Validated  47 At5g61430UCUACGUGCCC~UGCUUCUCCA Validated  48 At5g39610 CUCACGUGACC~UGCUUCUCCGPredicted  49 OsNAC1 CGCACGUGACC~UGCUUCUCCA Predicted  50 MtNACCUUACGUGUCC~UGCUUCUCCA Predicted  51 GmNAC CUUACGUGCCC~UGCUUCUCCAPredicted  52 LeNAC GCCACGUGCAC~UGCUUCUCCA Predicted  53miR165/166 family target sequences - all plants At1g30490UUGGGAUGAAG~CCUGGUCCGG Validated  54 At5g60690 CUGGGAUGAAG~CCUGGUCCGGValidated  55 At1g52150 CUGGAAUGAAG~CCUGGUCCGG Validated  56 PtHDZIPIIICCGGGAUGAAG~CCUGGUCCGG Predicted  57miR167 family target sequences - all plants At1g30330GAGAUCAGGCU~GGCAGCUUGU Validated  58 At5g37020 UAGAUCAGGCU~GGCAGCUUGUValidated  59 OsARF6 AAGAUCAGGCU~GGCAGCUUGU Predicted  60miR168 family target sequences - all plants At1g48410UUCCCGAGCUG~CAUCAAGCUA Validated  61miR169 family target sequences - all plants At1g17590AAGGGAAGUCA~UCCUUGGCUG Validated  62 At1g54160 ACGGGAAGUCA~UCCUUGGCUAValidated  63 At1g72830 AGGGGAAGUCA~UCCUUGGCUA Validated  64 At3g05690AGGCAAAUCAU~CUUUGGCUCA Validated  65 At3g20910 GCGGCAAUUCA~UUCUUGGCUUValidated  66 At5g12840 CCGGCAAAUCA~UUCUUGGCUU Predicted  67 At3g14020AAGGGAAGUCA~UCCUUGGCUA Predicted  68 ZmHAP2 GUGGCAACUCA~UCCUUGGCUCPredicted  69 VvHAP2 UGGGCAAUUCA~UCCUUGGCUU Predicted  70 OsHAP2AUGGCAAAUCA~UCCUUGGCUU Predicted  71 GmHAP2 UAGGGAAGUCA~UCCUUGGCUCPredicted  72 GhHAP2 CUGGGAAGUCA~UCCUUGGCUC Predicted  73miR170/171 family target sequences - all plants At2g45160GAUAUUGGCGC~GGCUCAAUCA Validated  74miR172 family target sequences - all plants At4g36920CUGCAGCAUCA~UCAGGAUUCU Validated  75 At2g28550 CAGCAGCAUCA~UCAGGAUUCUValidated  76 At5g60120 AUGCAGCAUCA~UCAGGAUUCU Validated  77 At5g67180UGGCAGCAUCA~UCAGGAUUCU Validated  78 At2g39250 UUGUAGCAUCA~UCAGGAUUCCPredicted  79 At3g54990 UUGCAGCAUCA~UCAGGAUUCC Predicted  80miR319 family target sequences - all plants At4g18390CAGGGGGACCC~UUCAGUCCAA Validated  81 At1g53230 GAGGGGUCCCC~UUCAGUCCAUValidated  82 At3g15030 GAGGGGUCCCC~UUCAGUCCAG Validated  83 At2g31070AAGGGGUACCC~UUCAGUCCAG Validated  84 At1g30210 UAGGGGGACCC~UUCAGUCCAAValidated  85 OsPCF5 GAGGGGACCCC~UUCAGUCCAG Predicted  86 OsPCF8UCGGGGCACAC~UUCAGUCCAA Predicted  87miR393 family target sequences - all plants At1g12820AAACAAUGCGA~UCCCUUUGGA Validated  88 At4g03190 AGACCAUGCGA~UCCCUUUGGAValidated  89 At3g23690 GGUCAGAGCGA~UCCCUUUGGC Validated  90 At3g62980AGACAAUGCGA~UCCCUUUGGA Validated  91miR394 family target sequences - all plants At1g27340GGAGGUUGACA~GAAUGCCAAA Validated  92miR395 family target sequences - all plants At5g43780GAGUUCCUCCA~AACACUUCAU Validated  93 At3g22890 GAGUUCCUCCA~AACUCUUCAUPredicted  94 At5g10180 AAGUUCUCCCA~AACACUUCAA Predicted  95miR396 family target sequences - all plants At2g22840UCGUUCAAGAA~AGCCUGUGGAA Validated  96 At2g36400 CCGUUCAAGAA~AGCCUGUGGAAValidated  97 At4g24150 UCGUUCAAGAA~AGCAUGUGGAA Validated  98 At2g45480ACGUUCAAGAA~AGCUUGUGGAA Validated  99 At3g52910 CCGUUCAAGAA~AGCCUGUGGAAPredicted 100 miR397 family target sequences - all plants At2g29130AAUCAAUGCUG~CACUCAAUGA Validated 101 At2g38080 AGUCAACGCUG~CACUUAAUGAValidated 102 At2g60020 AAUCAAUGCUG~CACUUAAUGA Validated 103miR398 family target sequences - all plants At1g08830AAGGGGUUUCC~UGAGAUCACA Validated 104 At2g28190 UGCGGGUGACC~UGGGAAACAUAValidated 105 At3g15640 AAGGUGUGACC~UGAGAAUCACA Validated 106miR173 family target sequences - Arabidopsis At1g50055GUGAUUUUUCUC~AACAAGCGAA Validated 107 At2g39675 GUGAUUUUUCUC~UACAAGCGAAValidated 108 At3g39680 GUGAUUUUUCUC~UCCAAGCGAA Validated 109miR399 family target sequences - all plants At2g33770UAGGGCAUAUC~UCCUUUGGCA Validated 110 At2g33770 UUGGGCAAAUC~UCCUUUGGCAValidated 111 At2g33770 UCGAGCAAAUC~UCCUUUGGCA Validated 112 At2g33770UAGAGCAAAUC~UCCUUUGGCA Validated 113 At2g33770 UAGGGCAAAUC~UUCUUUGGCAPredicted 114 OsE2UBC UAGGGCAAAUC~UCCUUUGGCA Predicted 115 OsE2UBCCUGGGCAAAUC~UCCUUUGGCA Predicted 116 OsE2UBC UCGGGCAAAUC~UCCUUUGGCAPredicted 117 OsE2UBC CCGGGCAAAUC~UCCUUUGGCA Predicted 118 PtE2UBCGCGGGCAAAUC~UUCUUUGGCA Predicted 119 MtE2UBC AAGGGCAAAUC~UCCUUUGGCAPredicted 120 TaE2UBC UAGGGCAAAUC~UCCUUUGGCG Predicted 121 TaE2UBCCUGGGCAAAUC~UCCUUUGGCG Predicted 122 TaE2UBC UUCGGCAAAUC~UCCUUUGGCAPredicted 123 miR403 family target sequences - dicots At1g31280GGAGUUUGUGC~GUGAAUCUAAU Validated 124miR390 family target sequences - all plants At3g17185CUUGUCUAUCCC~UCCUGAGCUA Validated 125 SbTAS3 UAUGUCUAUCCC~UUCUGAGCUGPredicted 126 SoTAS3 UAUGUCUAUCCC~UUCUGAGCUA Predicted 127 ZmTAS3aUAUGUCUAUCCC~UUCUGAGCUG Predicted 128 OsTAS3 UCGGUCUAUCCC~UCCUGAGCUGPredicted 129 PgTAS3 UUAGUCUAUCCC~UCCUGAGCUA Predicted 130 VvTAS3AUUGCCUAUCCC~UCCUGAGCUG Predicted 131 TcTAS3 CCUUGCUAUCCC~UCCUGAGCUGPredicted 132 LeTAS3 CUUGUCUAUCCC~UCCUGAGCUG Predicted 133 ZmTAS3bCCCUUCUAUCCC~UCCUGAGCUA Predicted 134 PtTAS3 CUUGUCUAUCCC~UCCUGAGCUAPredicted 135 OsTAS3b CCCUUCUAUCCC~UCCUGAGCUA Predicted 136 TaTAS3CCCUUCUAUCCC~UCCUGAGCUA Predicted 137 HvTAS3 CCUUUCUAUCCC~UCCUGAGCUAPredicted 138 PtTAS3b CCUGUCUAUCCC~UCCUGAGCUA Predicted 139 McTAS3UGUGUCUAUCCC~UCCUGAGCUA Predicted 140miR447 family target sequences - Arabidopsis At5g60760UGACAAACAUC~UCGUCCCCAA Validated 141 At3g45090 UGACAAACAUC~UCGUUCCUAAPredicted 142 miR408 family target sequences - all plants At2g02850CCAAGGGAAGA~GGCAGUGCAU Predicted 281 At2g30210 ACCAGUGAAGA~GGCUGUGCAGValidated 282 At2g47020 GCCAGGGAAGA~GGCAGUGCAU Predicted 283 At5g05390GCCGGUGAAGA~GGCUGUGCAA Predicted 284 At5g07130 GCCGGUGAAGA~GGCUGUGCAGPredicted 285

Between Jan. 7, 2005 and Jan. 7, 2006, the following changes were madeto nomenclature related to nuclei acid molecules described herein:

Systematic Names Assigned to TAS Loci by the Arabidopsis InformationResource (TAIR)

At2g39680 antisense (TAS1511) has become At2g39681 (TAS2)

AU235820 (TAS255a) has become At1g50055 (TAS1b)

CD534192 (TAS255b) has become At2g27400 (TAS1a)

TAS255c has become At2g39675 (TAS1c)

At3g17185 (ASR) has become At3g17185 (TAS3)

Official miRNA Name Assigned by the miRNA Registry (miRBase)

ASRP1890 has become miR447

These nomenclature changes are reflected in this document.

Interfering with or inhibiting (expression of a target sequence): Thisphrase refers to the ability of a small RNA, such as an siRNA or amiRNA, or other molecule, to measurably reduce the expression and/orstability of molecules carrying the target sequence. A target sequencecan include a DNA sequence, such as a gene or the promoter region of agene, or an RNA sequence, such as an mRNA. “Interfering with orinhibiting” expression contemplates reduction of the end-product of thegene or sequence, e.g., the expression or function of the encodedprotein or a protein, nucleic acid, other biomolecule, or biologicalfunction influenced by the target sequence, and thus includes reductionin the amount or longevity of the mRNA transcript or other targetsequence. In some embodiments, the small RNA or other molecule guideschromatin modifications which inhibit the expression of a targetsequence. It is understood that the phrase is relative, and does notrequire absolute inhibition (suppression) of the sequence. Thus, incertain embodiments, interfering with or inhibiting expression of atarget sequence requires that, following application of the small RNA orother molecule (such as a vector or other construct encoding one or moresmall RNAs), the sequence is expressed at least 5% less than prior toapplication, at least 10% less, at least 15% less, at least 20% less, atleast 25% less, or even more reduced. Thus, in some particularembodiments, application of a small RNA or other molecule reducesexpression of the target sequence by about 30%, about 40%, about 50%,about 60%, or more. In specific examples, where the small RNA or othermolecule is particularly effective, expression is reduced by 70%, 80%,85%, 90%, 95%, or even more.

Isolated: A biological component (such as a nucleic acid molecule,protein or organelle) that has been substantially separated or purifiedaway from other biological components in the cell of the organism inwhich the component naturally occurs, i.e., other chromosomal andextra-chromosomal DNA and RNA, proteins and organelles. Nucleic acidsand proteins that have been isolated include nucleic acids and proteinspurified by standard purification methods. The term also embracesnucleic acids and proteins prepared by recombinant expression in a hostcell as well as chemically synthesized nucleic acids.

MicroRNA (miRNA): Small, non-coding RNA gene products of approximately21 nucleotides long and found in diverse organisms, including animalsand plants. miRNAs structurally resemble siRNAs except that they arisefrom structured, foldback-forming precursor transcripts derived frommiRNA genes. Primary transcripts of miRNA genes form hairpin structuresthat are processed by the multidomain RNaseIII-like nuclease DICER andDROSHA (in animals) or DICER-LIKE1 (DCL1; in plants) to yield miRNAduplexes. The mature miRNA is incorporated into RISC complexes afterduplex unwinding. Plant miRNAs interact with their RNA targets withperfect or near perfect complementarity.

Mutation: A heritable change in DNA sequence. Mutations include aframe-shift, a point mutation, a missense mutation, a silent mutation, apolymorphism, a nonsense mutation, a deletion, a null mutation, atruncation, an elongation, an amino acid substitution, or an insertion.A mutant is an organism or cell carrying a mutation. The mutant can begenetically engineered or produced naturally.

Nucleotide: “Nucleotide” includes, but is not limited to, a monomer thatincludes a base linked to a sugar, such as a pyrimidine, purine orsynthetic analogs thereof, or a base linked to an amino acid, as in apeptide nucleic acid (PNA). A nucleotide is one monomer in anoligonucleotide/polynucleotide. A nucleotide sequence refers to thesequence of bases in an oligonucleotide/polynucleotide.

The major nucleotides of DNA are deoxyadenosine 5′-triphosphate (dATP orA), deoxyguanosine 5′-triphosphate (dGTP or G), deoxycytidine5′-triphosphate (dCTP or C) and deoxythymidine 5′-triphosphate (dTTP orT). The major nucleotides of RNA are adenosine 5′-triphosphate (ATP orA), guanosine 5′-triphosphate (GTP or G), cytidine 5′-triphosphate (CTPor C) and uridine 5′-triphosphate (UTP or U). Inosine is also a basethat can be integrated into DNA or RNA in a nucleotide (dITP or ITP,respectively).

Oligonucleotide: An oligonucleotide is a plurality of nucleotides joinedby phosphodiester bonds, between about 6 and about 300 nucleotides inlength. An oligonucleotide analog refers to compounds that functionsimilarly to oligonucleotides but have non-naturally occurring portions.For example, oligonucleotide analogs can contain non-naturally occurringportions, such as altered sugar moieties or inter-sugar linkages, suchas a phosphorothioate oligodeoxynucleotide. Functional analogs ofnaturally occurring polynucleotides can bind to RNA or DNA

Operably linked. A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame. In specific embodiments, operably linked nucleic acids asdiscussed herein are aligned in a linear concatamer capable of being cutinto 21-mer fragments, at least one of which is a siRNA.

Ornamental plant: A plant that is grown for visual display. Numerousplants are commonly recognized as ornamental. These include, forexample, indoor or outdoor nursery plants, house and garden plants, andflorist crops, each of which may include without limitation trees,shrubs, perennials, bulbs, annuals, groundcovers, turf grasses, herbs,or native plants.

Ortholog: Two nucleic acid or amino acid sequences are orthologs of eachother if they share a common ancestral sequence and diverged when aspecies carrying that ancestral sequence split into two species.Orthologous sequences are also homologous sequences.

Polymerization: Synthesis of a nucleic acid chain (oligonucleotide orpolynucleotide) by adding nucleotides to the hydroxyl group at the3′-end of a pre-existing RNA or DNA primer using a pre-existing DNAstrand as the template. Polymerization usually is mediated by an enzymesuch as a DNA or RNA polymerase. Specific examples of polymerasesinclude the large proteolytic fragment of the DNA polymerase I of thebacterium E. coli (usually referred to as Kleenex polymerase), E. coliDNA polymerase I, and bacteriophage T7 DNA polymerase. Polymerization ofa DNA strand complementary to an RNA template (e.g., a cDNAcomplementary to a mRNA) can be carried out using reverse transcriptase(in a reverse transcription reaction).

For in vitro polymerization reactions, it is necessary to provide to theassay mixture an amount of required cofactors such as M⁺⁺, and dATP,dCTP, dGTP, dTTP, ATP, CTP, GTP, UTP, or other nucleoside triphosphates,in sufficient quantity to support the degree of polymerization desired.The amounts of deoxyribonucleotide triphosphates substrates required forpolymerizing reactions are well known to those of ordinary skill in theart. Nucleoside triphosphate analogues or modified nucleosidetriphosphates can be substituted or added to those specified above.

Polypeptide: A polymer in which the monomers are amino acid residueswhich are joined together through amide bonds. When the amino acids arealpha-amino acids, either the L-optical isomer or the D-optical isomercan be used, the L-isomers being preferred. The term polypeptide orprotein as used herein encompasses any amino acid sequence and includesmodified sequences such as glycoproteins. The term polypeptide isspecifically intended to cover naturally occurring proteins, as well asthose that are recombinantly or synthetically produced.

Post-Transcriptional Gene Silencing (PTGS): A form of gene silencing inwhich the inhibitory mechanism occurs after transcription. This canresult in either decreased steady-state level of a specific RNA targetor inhibition of translation (Tuschl, ChemBiochem, 2:239-245, 2001). Inthe literature, the terms RNA interference (RNAi) andposttranscriptional cosuppression are often used to indicateposttranscriptional gene silencing.

Primer: Primers are relatively short nucleic acid molecules, usually DNAoligonucleotides six nucleotides or more in length. Primers can beannealed to a complementary target DNA strand (“priming”) by nucleicacid hybridization to form a hybrid between the primer and the targetDNA strand, and then the primer extended along the target DNA strand bya nucleic acid polymerase enzyme. Pairs of primers can be used foramplification of a nucleic acid sequence, e.g., by nucleic-acidamplification methods known in to those of ordinary skill in the art.

A primer is usually single stranded, which may increase the efficiencyof its annealing to a template and subsequent polymerization. However,primers also may be double stranded. A double stranded primer can betreated to separate the two strands, for instance before being used toprime a polymerization reaction (see for example, Nucleic AcidHybridization. A Practical Approach, Hames and Higgins, eds., IRL Press,Washington, 1985). By way of example, a double stranded primer can beheated to about 90°-100° C. for about 1 to 10 minutes.

Promoter: An array of nucleic acid control sequences which directtranscription of a nucleic acid. A promoter includes necessary nucleicacid sequences near the start site of transcription, such as, in thecase of an RNA polymerase II type promoter, a TATA element. Optionally,a promoter may include an enhancer and/or a repressor element. Enhancerand repressor elements can be located adjacent to, or distal to thepromoter, and can be located as much as several thousand base pairs fromthe start site of transcription. Representative examples of promotersthat can be used in the present disclosure are described herein.

Protein: A biological molecule, for example a polypeptide, expressed bya gene and comprised of amino acids.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified proteinpreparation is one in which the protein referred to is more pure (hasfewer impurities) than the protein in its natural environment within acell.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination can be accomplished by chemical synthesis orby the artificial manipulation of isolated segments of nucleic acids,e.g., by genetic engineering techniques.

Regulatable promoter: A promoter whose activity is regulated by anagent, such as a transcription factor, a chemical compound, or a nucleicacid molecule.

Regulating gene expression: The process of controlling the expression ofa gene by increasing or decreasing the expression, production, oractivity of an agent that affects gene expression. The agent can be aprotein, such as a transcription factor, or a nucleic acid molecule,such as a miRNA or an siRNA molecule, which when in contact with thegene or its upstream regulatory sequences, or a mRNA encoded by thegene, either increases or decreases gene expression.

RNA: A typically linear polymer of ribonucleic acid monomers, linked byphosphodiester bonds. Naturally occurring RNA molecules fall into threegeneral classes, messenger (mRNA, which encodes proteins), ribosomal(rRNA, components of ribosomes), and transfer (tRNA, moleculesresponsible for transferring amino acid monomers to the ribosome duringprotein synthesis). Messenger RNA includes heteronuclear (hnRNA) andmembrane-associated polysomal RNA (attached to the rough endoplasmicreticulum). Total RNA refers to a heterogeneous mixture of all types ofRNA molecules.

RNA-dependent RNA polymerase (RDR): Enzyme that polymerizes formation ofRNA using a single-stranded RNA template. This frequently results information of a double-stranded RNA molecule. Examples of ArabidopsisRDRs include RDR1, RDR2 and RDR6 (Xie et al., PLoS Biol 2:642-652,2004). RDRs required for viral replication are also encoded by manyviruses (Kao et al., Virology 287:251-260, 2001).

RNA interference (RNAi): Gene silencing mechanisms that involve smallRNAs (including miRNA and siRNA) are frequently referred to under thebroad term RNAi. Natural functions of RNAi include protection of thegenome against invasion by mobile genetic elements such as transposonsand viruses, and regulation of gene expression.

RNA interference results in the inactivation or suppression ofexpression of a gene within an organism. RNAi can be triggered by one oftwo general routes. First, it can be triggered by direct cellulardelivery of short-interfering RNAs (siRNAs, usually ˜21 nucleotides inlength and delivered in a dsRNA duplex form with two unpairednucleotides at each 3′ end), which have sequence complementarity to aRNA that is the target for suppression. Second, RNAi can be triggered byone of several methods in which siRNAs are formed in vivo from varioustypes of designed, expressed genes. These genes typically express RNAmolecules that form intra- or inter-molecular duplexes (dsRNA) which areprocessed by natural enzymes (DICER or DCL) to form siRNAs. In somecases, these genes express “hairpin”-forming RNA transcripts withperfect or near-perfect base-pairing; some of the imperfecthairpin-forming transcripts yield a special type of small RNA, termedmicroRNA (miRNA). In either general method, it is the siRNAs (or miRNAs)that function as “guide sequences” to direct an RNA-degrading enzyme(termed RISC) to cleave or silence the target RNA. In some cases, it isbeneficial to integrate an RNAi-inducing gene into the genome of atransgenic organism. An example would be a plant that is modified tosuppress a specific gene by an RNAi-inducing transgene. In most methodsthat are currently in practice, RNAi is triggered in transgenic plantsby transgenes that express a dsRNA (either intramolecular or hairpin, orintermolecular in which two transcripts anneal to form dsRNA).

RNA silencing: A general term that is used to indicate RNA-based genesilencing or RNAi.

Sequence identity: The similarity between two (or more) nucleic acidsequences, or two (or more) amino acid sequences, is expressed in termsof the similarity between the sequences, otherwise referred to assequence identity or homology. Sequence identity is frequently measuredin terms of percentage identity (or similarity or homology); the higherthe percentage, the more similar the two sequences are. Homologs ororthologs of a specified protein, and the corresponding cDNA sequence,will possess a relatively high degree of sequence identity when alignedusing standard methods. This homology will be more significant when theorthologous proteins or cDNAs are derived from species which are moreclosely related (e.g., different plant sequences), compared to speciesmore distantly related (e.g., human and Arabidopsis sequences).

Typically, orthologs are at least 50%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 93%, atleast 95%, or at least 98% identical at the nucleotide level and atleast 50%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 93%, at least 95%, or at least 98%identical at the amino acid level when comparing a protein to anorthologous protein.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman Adv. Appl. Math. 2: 482, 1981; Needleman & Wunsch J. Mol. Biol.48: 443, 1970; Pearson & Lipman Proc. Natl. Acad. Sci. USA 85: 2444,1988; Higgins & Sharp Gene, 73: 237-244, 1988; Higgins & Sharp CABIOS 5:151-153, 1989; Corpet et al. Nuc. Acids Res. 16:10881-10890, 1988; Huanget al. Computer Appls. Biosciences 8:155-165, 1992; and Pearson et al.Meth. Mol. Bio. 24:307-331, 1994. Altschul et al. (J. Mol. Biol.215:403-410, 1990) present a detailed consideration of sequencealignment methods and homology calculations. Multiple sequences can bealigned, for instance, using programs such as CLUSTAL-W or TCoffee.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al. J.Mol. Biol. 215:403-410, 1990) is available from several sources,including the National Center for Biotechnology Information (NCBI,Bethesda, Md.) and on the Internet, for use in connection with thesequence analysis programs blastp, blastn, blastx, tblastn and tblastx.It can be accessed at the NCBI website, together with a description ofhow to determine sequence identity using this program.

For comparisons of amino acid sequences of greater than about 30 aminoacids, the Blast 2 sequences function is employed using the defaultBLOSUM62 matrix set to default parameters, (gap existence cost of 11,and a per residue gap cost of 1). When aligning short peptides (fewerthan around 30 amino acids), the alignment should be performed using theBlast 2 sequences function, employing the PAM30 matrix set to defaultparameters (open gap 9, extension gap 1 penalties). Proteins with evengreater similarity to the reference sequence will show increasingpercentage identities when assessed by this method, such as at least70%, at least 75%, at least 80%, at least 90%, at least 91%, at least92%, at least 93%, at least 94% or at least 95% sequence identity. Whenless than the entire sequence is being compared for sequence identity,homologs will typically possess at least 75% sequence identity overshort windows of 10-20 amino acids, and may possess sequence identitiesof at least 85% or at least 90% or 95% or more depending on theirsimilarity to the reference sequence. Methods for determining sequenceidentity over such short windows are described at the NCBI web-site,frequently asked questions (FAQ) page. One of ordinary skill in the artwill appreciate that these sequence identity ranges are provided forguidance only; it is entirely possible that strongly significanthomologs could be obtained that fall outside of the ranges provided.

An alternative indication that two nucleic acid molecules are closelyrelated is that the two molecules hybridize to each other understringent conditions. Stringent conditions are sequence-dependent andare different under different environmental parameters. Generally,stringent conditions are selected to be about 5° C. to 20° C. lower thanthe thermal melting point (T_(m)) for the specific sequence at a definedionic strength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence remains hybridizedto a perfectly matched probe or complementary strand. Conditions fornucleic acid hybridization and calculation of stringencies can be foundin Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, CSHL,New York and Tijssen (1993) Laboratory Techniques in Biochemistry andMolecular Biology-Hybridization with Nucleic Acid Probes Part I, Chapter2, Elsevier, New York. Nucleic acid molecules that hybridize understringent conditions to a human p28ING5 gene sequence will typicallyhybridize to a probe based on either an entire human p28ING5 gene orselected portions of the gene under wash conditions of 2×SSC at 50° C.

Nucleic acid sequences that do not show a high degree of identity cannevertheless encode similar amino acid sequences, due to the degeneracyof the genetic code. It is understood that changes in nucleic acidsequence can be made using this degeneracy to produce multiple nucleicacid molecules that all encode substantially the same protein.

Silencing agent or molecule: A specific molecule, which can exert aninfluence on a cell in a sequence-specific manner to reduce or silencethe expression or function of a target, such as a target gene orprotein. Examples of silence agents include nucleic acid molecules suchas naturally occurring or synthetically generated small interfering RNAs(siRNAs), naturally occurring or synthetically generated microRNAs(miRNAs), naturally occurring or synthetically generated dsRNAs, andantisense sequences (including antisense oligonucleotides, hairpinstructures, and antisense expression vectors), as well as constructsthat code for any one of such molecules.

Specific binding agent: An agent that binds substantially only to adefined target. Thus a protein-specific binding agent bindssubstantially only the specified protein.

Small interfering RNA (siRNA): RNA of approximately 21-25 nucleotidesthat is processed from a dsRNA by a DICER enzyme (in animals) or a DCLenzyme (in plants). The initial DICER or DCL products aredouble-stranded, in which the two strands are typically 21-25nucleotides in length and contain two unpaired bases at each 3′ end. Theindividual strands within the double stranded siRNA structure areseparated, and typically one of the siRNAs then are associated with amulti-subunit complex, the RNAi-induced silencing complex (RISC). Atypical function of the siRNA is to guide RISC to the target based onbase-pair complementarity.

Target nucleic acid (to be inhibited): Any nucleic acid containing asequence that interacts with a miRNA or siRNA, or that has the potentialto yield a sequence that interacts with a miRNA or siRNA (for example,through transcription of a locus). The target can be a cellular nucleicacid, such as a mRNA that encodes an essential or non-essential protein,or a foreign nucleic acid, such as a virus-derived or transgene-derivedRNA molecule. The target can be a DNA sequence corresponding to apromoter, or a sequence corresponding to any expressed region of agenome, for instance.

Trans-acting siRNAs: A subclass of siRNAs that function like miRNAs torepress expression of target genes, yet have unique biogenesisrequirements. Trans-acting siRNAs form by transcription ofta-siRNA-generating genes, cleavage of the transcript through a guidedRISC mechanism, conversion of one of the cleavage products to dsRNA, andprocessing of the dsRNA by DCL enzymes. ta-siRNAs are unlikely to bepredicted by computational methods used to identify miRNA because theyfail to form a stable foldback structure. Data provided hereindemonstrate that ta-siRNAs are not an Arabidopsis oddity, but areconserved among distantly related plant species and have been maintainedover a long evolutionary period.

A ta-siRNA precursor is any nucleic acid molecule, includingsingle-stranded or double-stranded DNA or RNA, that can be transcribedand/or processed to release a ta-siRNA.

Transcriptional gene silencing (TGS): A phenomenon that is triggered bythe formation of dsRNA that is homologous with gene promoter regions andsometimes coding regions. TGS results in DNA and histone methylation andchromatin remodeling, thereby causing transcriptional inhibition ratherthan RNA degradation. Both TGS and PTGS depend on dsRNA, which iscleaved into small (21-25 nucleotides) interfering RNAs (Eckhardt, PlantCell, 14:1433-1436, 2002; Aufsatz et al., Proc. Natl. Acad. Sci. U.S.A.,99:16499-16506, 2002).

Transgenic (plant/fungus/cell/other entity): This term refers to aplant/fungus/cell/other entity that contains recombinant geneticmaterial not normally found in entities of this type and which has beenintroduced into the entity in question (or into progenitors of theentity) by human manipulation. Thus, a plant that is grown from a plantcell into which recombinant DNA is introduced by transformation is atransgenic plant, as are all offspring of that plant that contain theintroduced transgene (whether produced sexually or asexually).

Triggering RNA: RNA transcript of an siRNA generating locus which isconverted into a dsRNA molecule by an RNA-dependent RNA polymerase (RDR)in vivo.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. Hence “comprisingA or B” means including A, or B, or A and B. It is further to beunderstood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

III. Overview of Several Embodiments

An siRNA-triggering or RNAi-triggering nucleic acid cassette isprovided, which cassette comprises an initiator sequence consisting ofabout 20 to 25 nucleotides, the initiator sequence having an initiationcleavage site between the tenth and eleventh nucleotide counted from the3′ end of the initiator sequence; and at least one gene suppressingsegment in about 21-nucleotide register (or phase) counted eitherupstream or downstream from the initiation cleavage site, wherein thegene suppressing segment or its complement is substantiallycomplementary to an RNA transcribed from a target gene selected forsiRNA inhibition. Also provided are expression vectors which include atleast one such nucleic acid cassette operably linked to a promoter.

Specific example initiator sequences are provided herein, for instance,in SEQ ID NOs: 1-142 and 281-285.

Also provided are siRNA- or RNAi-triggering nucleic acids (bothcassettes and vectors) that comprise two or more gene suppressingsegments. In embodiments having two or more gene suppressing segments,these segments optionally can be directed to (complementary with) two ormore different genes or other target sequences selected for siRNAinhibition.

Cells and organisms into which have been introduced a vector or cassetteof this disclosure are also provided, as are parts of multicellularorganisms that contain such transgenic nucleic acids. Thus, anotherspecific embodiment is a seed for a transgenic plant that expresses RNAfor suppressing a target gene, wherein said seed and plant compriserecombinant DNA from which there is transcribed a first RNA comprisingan initiator segment consisting of 20-25 nucleotides wherein aninitiation cleavage site is located between the tenth and eleventhnucleotide counted from the 3′ end of the initiator segment and whereinsaid initiator segment is linked to or overlaps with at least one genesuppressing segment of 21-nucleotides in precise 21-nucleotide registercounted either upstream or downstream from the initiation cleavage site,wherein said gene suppressing segment or it complement is complementaryto mRNA transcribed from said target gene.

Yet other embodiments are seed for a transgenic plant further comprisingDNA from which there is transcribed a second RNA that hybridizes to saidfirst RNA at said initiation cleavage site. By way of example, thesecond RNA in some instances is an exogenous miRNA, or a miRNAtranscribed from a native plant gene or a heterologous gene or any genenot native to the plant. Seed are also provided for a transgenic plantwherein the first RNA comprises two or more gene suppressing segments.

Optionally, the target gene in any provided organism can be endogenousto that organism. For instance, the target gene may be an endogenousplant gene, an endogenous fungal gene, or an endogenous invertebrategene, in plant, fungal, or invertebrate embodiments, respectively. Thereis also therefore provided a seed for a transgenic plant of thedisclosure, wherein the plant is corn and the endogenous plant geneencodes lysine ketoglutarate reductase.

Alternatively, the target gene could be exogenous to the transgenicorganism, for instance it could be a gene of a pathogen or a pest, suchas a plant pathogen or plant pest. In specific examples, such plant pestis a nematode or insect or such pant pathogen is a virus or fungus. Inone particular embodiment, a seed for a transgenic plant is providedwherein said plant is soybean and said plant pest is soybean cystnematode.

In yet another provided seed for a transgenic plant of the disclosure,the recombinant DNA comprises a promoter functional in said plant andoperably linked to DNA coding for the first RNA. Such a promoter in somecases is characterized as being a constitutive promoter, an induciblepromoter, a tissue specific promoter, a ubiquitous promoter or acombination thereof.

Also provided are seed for a transgenic plant as described, wherein theplant is a corn, soybean, cotton, canola, wheat or rice plant.

Optionally, in any of the provided embodiments of seed for a transgenicplant, the recombinant DNA further comprises nucleotides for expressingat least one protein.

Also provided herein are methods of inhibiting expression of a targetgene in a cell, the method comprising exposing the cell to an effectiveamount of a RNAi-triggering or siRNA-triggering nucleic acid cassette ora vector as described. The cell can be, for instance, a plant cell, afungal cell, or an invertebrate cell. It is particularly contemplatedthat the cell could be in vitro or in vivo, for instance, contained in amulticellular organism.

Yet another method is provided, which is a method of inducing productionof at least one siRNA in a cell. This method involves transforming thecell with a recombinant nucleic acid molecule comprising a nucleic acidcassette as described herein, wherein the recombinant nucleic acidmolecule directs expression of a mRNA from the nucleic acid cassette,which mRNA is processed in the cell to produce at least one siRNA,thereby inducing the production of at least one siRNA in the cell.

Another method is provided, which is a method of inhibiting activity ofa target gene in a plant cell. This method involves transforming theplant cell with a recombinant nucleic acid molecule comprising a nucleicacid cassette as described herein, wherein at least one gene suppressingsegment of the nucleic acid cassette is specific for the target gene;and expressing the nucleic acid molecule, thereby producing in the plantcell at least one siRNA specific for the target gene which inhibitsactivity of the target gene in the plant cell.

Another method is a method of inhibiting activity of a target gene in aplant seed, comprising providing in cells of said plant a recombinantnucleic acid molecule comprising a nucleic acid cassette of thedisclosure, wherein at least one gene suppressing segment of the nucleicacid cassette is specific for the target gene and wherein said cassettecomprises a seed-specific promoter operably linked to said initiatorsequence and said at least one gene suppressing segment; a recombinantDNA with a seed specific promoter operably linked to DNA transcribing anmiRNA that hybridizes to said initiator sequence at said initiationcleavage site; both.

IV. Methods of Triggering RNA Interference (RNAi)

Plants and animals use small RNAs [microRNAs (miRNAs) and siRNAs] asguides for posttranscriptional and epigenetic regulation of targetgenes. In plants, miRNAs and trans-acting (ta) siRNAs form throughdistinct biogenesis pathways, although they both interact with targettranscripts and guide cleavage. An integrated approach to identifytargets of Arabidopsis thaliana miRNAs and ta-siRNAs revealed severalnew classes of small RNA-regulated genes. These included conventionalgenes, such as the RNAi factor Argonaute2 (miR403), an E2-ubiquitinconjugating enzyme (miR399), and two Auxin Response Factors (TAS3ta-siRNAs). Five ta-siRNA-generating transcripts were identified astargets of miR173 or miR390. Rather than functioning to negativelyregulate these transcripts, miR173- and miR390-guided cleavage was shownto set the 21-nucleotide phasing for ta-siRNA precursor processing.These data support a model in which miRNA-guided formation of a 5′ or 3′terminus within pre-ta-siRNA transcripts, followed by RDR6-dependentformation of dsRNA and DCL1-mediated processing, yields phased ta-siRNAsthat negatively regulate other genes.

In Example 1, new Arabidopsis miRNA and ta-siRNA targets are identifiedthrough an integrated strategy that included computational, genome-wideexpression profiling and experimental validation components. Throughidentification of genes significantly upregulated in miRNA or ta-siRNAbiogenesis mutants (hyl1-2, hst-15, dcl1-7, hen1-1, and rdr6-2) usingmicroarrays, data is presented herein that demonstrates identificationof genes potentially regulated by miRNAs and ta-siRNAs. Two genes, ARF3and ARF4, were found to contain a duplicated conserved 21 sequence.Analysis of an Arabidopsis sequence, conserved across angiosperms,identified small RNAs typical of ta-siRNAs that could target ARF3 andARF4 mRNAs.

As taught herein, RNAi can be induced using transgenes or otherdelivered genes or constructs that encode non-dsRNA-forming transcripts.This method exploits the occurrence of natural siRNAs and miRNAs thatcan: 1) interact with the delivered transcript through base-pairing, 2)engage a natural dsRNA-forming enzyme termed an RNA-dependent RNApolymerase (RDR), and 3) engage natural DICER-LIKE (e.g., DCL1) enzymesto form siRNAs in precise and predictable register. The siRNAs that formunder this mechanism can function to suppress target mRNA expression ifthe target contains a high degree of sequence complementarity to thesiRNAs. One advantage of this method is that it circumvents the need todeliver a dsRNA-forming entity or transgene to initiate the RNAi processof gene suppression.

The methods described herein also enable RNAi to target multiple mRNAsor other target RNAs, depending on the specific siRNA units designedinto the construct. The method also permits highly specific siRNAformation rather than non-specific siRNA formation (which results in anincreased chance of off-target effects) using conventional dsRNA-formingconstructs. The method also may take advantage of naturally occurringmiRNAs and siRNAs with tissue- or cell-specific expressioncharacteristics to drive tissue- and cell-specificity of RNAi.Alternatively, a heterologous miRNA or siRNA can be added to the cell(for instance by providing an expression cassette encoding suchmolecule) in order to provide the receptive element necessary to mediatecleavage and release of siRNAs from a RNAi-triggering nucleic acidcassette.

Also provided herein are nucleic acid constructs that generate, in vivo,siRNAs useful for triggering RNAi-like responses. Representative methodsfor producing such constructs, as well as guidelines for selectingelements included therein, are provided.

V. Initiator Sequences and Identification Thereof

When present in an RNA molecule, an initiator sequence serves as a sitethat interacts with a miRNA or siRNA, which guides cleavage through theactivity of RISC. Cleavage at an initiator sequence cleavage site(usually between the tenth and eleventh nucleotide counting from the 3′end of the initiator sequence) sets the 21-nucleotide register withinthe RNA molecule, resulting in additional cleavages of the RNA moleculeby the Dicer or DCL protein at usually 21-nucleotide intervals upstreamand/or downstream of the initiator sequence. In an engineeredRNAi-triggering nucleic acid cassette as described herein, suchadditional, in-phase cleavages release siRNAs from RNA molecules thatare transcribed from the cassette. Representative initiator sequences inRNA form, also referred to as miRNA target sequences, are shown in SEQID NOs: 1-142 and 281-285.

Any sequence in an RNA molecule to which a siRNA or miRNA can bind bycomplementarity, or any sequence in a DNA molecule that encodes for sucha sequence in an RNA molecule, can serve as an initiator sequence. Inaddition to representative initiator sequences provided herein, methodsare provided for identifying additional sequences from other genes,other plant species, or any other organisms. An integrated system isprovided herein for identifying new miRNA and ta-siRNA targets. Thissystem involves computational, genome-wide expression profiling andexperimental validation components. As demonstrated in Example 1, thesystem reliably identifies prospective initiator sequences, which aretarget sites for miRNAs. Representative initiator sequences, includingmany identified and validated using the computational system provided,are shown in SEQ ID NO: 1-142 and 281-285.

In general, an initial pool of predicted target sites for validatedmiRNAs was created by FASTA searches using a +15/−10 scoring matrix ofthe TAIR AGI transcript database, limited to 4 mispairs, 4 G:U pairs, toa total of seven, with 100,000 results obtained for the reversecomplement of each small RNA. A single, one nucleotide gap was allowed.In the embodiment described in Example 1, the miRNA target predictionalgorithm used to score these sites was developed based on 94experimentally validated and predicted family members of miRNA-targetsite duplexes, including 66 targets validated in previous studies plus28 family members with conserved miRNA target sites (Target Rule Set).

Three filters based on the Target Rule Set were applied sequentially. Ineach case, base one was considered to be the first nucleotide from the5′ end of the miRNA. First, targets with a mismatch score greater thanfour were excluded. The Minimum Free Energy (ΔG_(MFE)) of a perfectmiRNA-target duplex was determined by computationally attaching aperfectly complementary target sequence to a small RNA using a four base“gap” linker sequence (----). The free energy each miRNA-predictedtarget site (ΔG_(target)) was determined by computationally linking thetarget sequence to the small RNA, from which the MFE ratio wascalculated (ΔG_(target)/ΔG_(MFE)). All thermodynamic values werecalculated using RNAFold in the Vienna RNA package. Remaining targetswith an MFE ratio less than 0.73 were excluded. Conservation of thetarget sequence was determined by using the region containing the targetsequence in a BLAST search against target transcripts, for instance, theArabidopsis transcript and EST databases, NCBI EST database, and O.sativa Unigene database in Example 1, and removing any targets with nomatches with less than three base changes in the target sequence.Duplicate target sites (identical genes) for related miRNA familymembers were combined in the final target gene set.

VI. Selection of Initiation Sequence for RNAi-Triggering Constructs

Any nucleic acid sequence that will serve to mediate cleavage by amiRNA- or siRNA-guidedRISC mechanism may be used as the initiatorsequence in constructs provided herein. Examples of such sequences areprovided herein, for instance in SEQ ID NOs: 1-142 and 281-285. It isnoted that the presented sequences are RNA sequences. It will beapparent to one of ordinary skill in the art that DNA constructs, suchas DNA constructs used in transformation of target cells, will containthe DNA equivalent of the listed RNA sequences.

By way of example, SEQ ID NO: 1 is GUGCUCUCUCUCUUCUGUCA (shown 5′ to3′). The corresponding miRNA sequence (also shown 5′ to 3′) isUGACAGAAGAGAGUGAGCAC (SEQ ID NO: 155); this is the reverse complement ofthe target/initiation sequence shown in SEQ ID NO: 1. A DNA constructcontaining an initiator sequence corresponding to SEQ ID NO: 1 wouldinclude the following sequence: 5′-GTGCTCTCTCTCTTCTGTCA-3′ (SEQ ID NO:280), which may be generated in double-stranded format depending on theembodiment. In such a DNA construct, the transcription site andstrandedness would be designed so the initiator sequence is produced asshown in SEQ ID NO: 1. This enables the native or provided,corresponding miRNA to bind by complementarity to the initiatorsequence.

It is noted that, in many embodiments, the initiator sequence and afirst gene suppressing element may overlap. This arises because theregister that is set by the initiator cleavage site begins at that site.Thus, the nucleotides of the 5′ or 3′ portion of the initiator sequencewill be incorporated into the first 21-mer gene suppressing element(e.g., siRNA) produced. This is illustrated, for instance, in FIGS.5A-C, FIG. 6A, and FIG. 7.

Many miRNAs and their corresponding target sequences (also referred toherein as initiator sequences) are highly conserved among distantlyrelated species. In plants in particular, target sequences that arerecognized by related miRNAs in different species differ only by one tothree bases, making computational prediction of target sites bysimilarity searches relatively straightforward (Jones-Rhoades & Bartel,Mol Cell 14:787-799, 2004). Owing to the high level of conservation ofmiRNAs, a functional miRNA target site from one plant species is likelyto be functional in a species which expresses the targeting miRNA. Forexample, miRNA target genes from Arabidopsis expressed in Nicotiana arecleaved by endogenous miRNAs (Llave et al., Science 297:2053-2056,2002). In Oryza and Populus, for which near-complete genomic sequenceinformation exists, homologous miRNA and/or target genes have beenidentified for 20 of 25 validated miRNA families in Arabidopsis. Forthese 20 conserved miRNA families, conserved homologous miRNA and/ortarget genes have also been found in several other plant species withless complete sequence information.

By way of example, in Table 2, miRNAs are grouped by related families(one to three nucleotide differences), or by targets of the miRNAfamily. Presence of the miRNA or target in a listed plant genus isindicated by an “X”. In generating this table, miRNA genes wereconsidered to be conserved if the homologous sequence was within 1-3nucleotides of the Arabidopsis sequence, formed a stable foldbackstructure, and did not encode an identifiable protein. Target sites wereconsidered to be conserved if the target gene in the specified genusencodes a protein similar to the Arabidopsis target gene.

TABLE 2 Conservation of miRNAs and target genes in plants. miR156/157miR158 miR159 miR160 miR161 miR162 miR163 miR164 miR165/166 miRNA miRNAmiRNA miRNA miRNA miRNA miRNA miRNA miRNA Genus Target Target TargetTarget Target Target Target Target Target Acorus X X Aegilops Allium XAmborella Antirrhinum X X X X X Apium Arabidopsis X X X X X X X X X X XX X X X X X Arachis Beta X Betula Brassica X X X Brugeria Capsicum X X XCeratopteris X X X Citrus X X Cryptomeria Cycas Descurainia EschscholziaX X Eucalyptus Glycine X X X X X X X X X X Gossypium X X X X Hedyotis XHelianthus X X Hordeum X X X X X Ipomoea X X X Lactuca X X X X LinumLiriodendron X X X X X Lotus X X X Lupinus X Lycopersicon X X X X XMalus Manihot Messmbryanthemum Medicago X X X X X X X X Nicotiana X X XX X X X Nuphar X Oryza X X X X X X X X X X X Pennisetum X PerseaPhaseolus X X Phycomitrella X X Picea Pinus X X X Poncirus X Populus X XX X X X X X X X X X Prunus X X X Robinia Rosa Saccharum X X X X X XSchedonorus X Sueada Secale X Sesamum X Solanum X X X X X Sorghum X X XX X X X X X Stevia Thellungiella Theobroma Triphysaria Triticum X X X XX X X X X Vitis X X X X X X Zea X X X X X X X Zinnia miR167 miR168miR169 miR170/171 miR172 miR173 miR319 miR390/391 miR393 miRNA miRNAmiRNA miRNA miRNA miRNA miRNA miRNA miRNA Genus Target Target TargetTarget Target Target Target Target Target Acorus X Aegilops X Allium X XAmborella Antirrhinum X X X X Apium Arabidopsis X X X X X X X X X X X XX X X X X X Arachis Beta Betula X Brassica X X X X Brugeria X X CapsicumX Ceratopteris X X X X X X Citrus X X X X Cryptomeria X CycasDescurainia X X Eschscholzia X Eucalyptus X Glycine X X X X X X X X X XX X X X Gossypium X X X X X X Hedyotis X X X Helianthus X X Hordeum X XX X X X X X X X Ipomoea X X Lactuca X X X Linum Liriodendron X X X X X XX Lotus X X X X X Lupinus X Lycopersicon X X X X X X X Malus X Manihot XX Messmbryanthemum X X Medicago X X X X X X X X X X Nicotiana X X X X XX X X Nuphar X Oryza X X X X X X X X X X X X X X Pennisetum Persea X X XPhaseolus X X Phycomitrella X X Picea X X Pinus X X X Poncirus X PopulusX X X X X X X X X X X X Prunus X X Robinia Rosa Saccharum X X X X X X XX X X X Schedonorus Sueada X Secale Sesamum Solanum X X X X X X X XSorghum X X X X X X X X X X X X X X Stevia Thellungiella Theobroma X X XX Triphysaria X Triticum X X X X X X X X X X X X X Vitis X X X X X X X XX X Zea X X X X X X X X X X X X X X Zinnia X X X miR394 miR395 miR396miR397 miR398 miR399 miR403 miRNA miRNA miRNA miRNA miRNA miRNA miRNAGenus Target Target Target Target Target Target Target Acorus X X XAegilops X Allium X X X Amborella X X Antirrhinum Apium X Arabidopsis XX X X X X X X X X X X X X Arachis X Beta X X X Betula Brassica X X X XBrugeria Capsicum X X X Ceratopteris Citrus X X X X Cryptomeria X XCycas X Descurainia Eschscholzia X X X Eucalyptus Glycine X X X X X X XX X Gossypium X X X X Hedyotis X X Helianthus X X X Hordeum X X X X XIpomoea X X Lactuca X X X X X Linum X Liriodendron X X X X Lotus X X X XX X Lupinus X Lycopersicon X X X X X Malus Manihot Messmbryanthemum XMedicago X X X X X X X X Nicotiana X X X X X X X Nuphar X Oryza X X X XX X X X Pennisetum X Persea Phaseolus X X Phycomitrella Picea X Pinus XX X Poncirus X Populus X X X X X X X X X X X X Prunus X X Robinia X RosaX Saccharum X X X X X Schedonorus Sueada Secale Sesamum X Solanum X X XX X X Sorghum X X X X X X X X X Stevia X Thellungiella X TheobromaTriphysaria X Triticum X X X X X X X X X Vitis X X X Zea X X X X X X X XZinnia X X

VII. Selection of Gene Suppressing Elements and Targets forRNAi-Triggering Constructs

A gene suppressing element is any nucleotide sequence which leads to thedownregulation of the final functional product of a gene, either RNA orprotein. For RNAi, this sequence is a 20 to 25 nucleotide RNA withcomplementarity to the gene to be suppressed.

Beneficial characteristics of a gene suppressing element useful forinclusion “in register” in an RNAi-triggering cassette are those knownto produce a functional (measurably effective for reducing expression ofa target gene/sequence) siRNA sequence. Empirical studies such asdescribed herein can be used to identify gene suppressing elements.There are also art-recognized guidelines that provide predictive RISCincorporation rules (Khvorova et al., Cell 115:209-216, 2003; Schwarz etal., Cell 115:199-208, 2003)

Specific gene suppressive elements can be designed depending on thetarget sequences (e.g., gene(s), regulatory sequence or invasive orpathogenic entities) to be suppressed. Gene suppressive elements(usually about 21-nucleotides in length), complementary to a target(e.g., gene transcript) to be suppressed, are included theRNAi-triggering cassette, in register, in either sense or antisenseorientation starting from the initiation cleavage site. At least eight,possibly more, unique (or duplicated) sequences can be included eitherupstream or downstream of the initiation cleavage site. Beyond theeighth register, processing by DICER or DCL enzymes may become lessprecise, and the 21 nucleotide register is more likely to becompromised. Even so, gene suppressive elements beyond eight can beoptionally included in constructs, including elements that are not inprecise 21-nucleotide register.

Gene suppressive elements contained in the RNAi-inducing cassette can bedesigned to target one or more genes, with one or more unique targetsequences. Potential targets might include, but are not limited to,pathogens, toxins, genes that lead to production of undesirable flavorsand/or odors, reproductive genes which could facilitate pollination orincrease crop yield, color or pigment genes, transcription factors,pathogen response genes, and genes involved in cold/water/drought andother environmental stresses. Related gene families, pathway-relatedgenes, or quantitative trait loci also may be targeted, for instance ina single RNAi-inducing cassette or a set of such cassettes. Suchfamily-directed cassettes are useful in the down-regulation of all (orselect) members of a gene family, all (or select) members in abiosynthetic pathway, and so forth, thereby yielding coordinateddownregulation of sets of genes.

Additional gene suppressive elements that are contemplated are directedto the genes of pathogens or pests associated with the resultant targetorganism; endogenous genes of the target organism that are involved inresponse to such pathogens or pests; and exogenous (heterologous)transgenes provided to the target organism (separately or in a singleconstruct containing the RNAi-triggering cassette) to influenceinfection or infestation or association of such pathogens or pests.

Gene suppressive elements also can be from any endogenous gene that itis desired to downregulate. Genes that negatively influence acharacteristic (that cause an unpleasant flavor, aroma, etc.) of thetarget organism; genes that lead to production of a toxin, allergen, orother detrimental component (e.g., erucic acid in an oil seed; hazardousallergens in peanuts; toxic compounds in potatoes, apricots); genesinvolved in reproduction (where inhibition will result in increasedvegetative production in a plant, for instance); genes involved in malefertility in plants (in order to produce male-sterile, non-selfingplants); genes that enhance vegetative growth (where reproductive growthis desired over seed production, such as in leaf crops like lettuce andspinach); genes that govern or influence color (for instance, the colorof leaves or bracts, flowers, stems, fruit, and so forth, where it isdesired to change the color); genes that govern or influencesusceptibility to stress (such as cold stress, water or drought stress,shear stress, and so forth); and transcription factors (where it isdesired to influence a downstream gene or set of genes the expression ofwhich is influenced by the transcription factor) are all examples ofconceived of targets for suppression using the methods and constructsdescribed herein.

It is further contemplated that transgenic plants produced using methodsand cassettes described herein can be further enhanced with stackedtraits, e.g. a crop having an enhanced agronomic trait resulting fromgene suppression from an siRNA-triggering nucleic acid cassette incombination with DNA expressing a protein supplementing the agronomictrait, or conferring another trait such as herbicide and/or pestresistance traits. For example, a trait can be enhanced by simultaneoussuppression of one gene and over expression of another gene to providetransgenic corn with an enhance level of the amino acid lysine.Transgenic corn with recombinant DNA for expression of the gene encodingdihydrodipicolinate synthase in the lysine synthetic pathway andsuppression of the gene encoding lysine ketoglutarate reductase (LKR) inthe lysine catabolic pathway has enhanced lysine as compared to controlplants. Following the methods of this disclosure, the suppression of LKRcan be effected by identifying a 21-nucleotide segment of the geneencoding LKR for insertion into an siRNA-triggering nucleic acidcassette. To effect the enhance lysine trait preferentially in seedtissue, seed specific promoters are used to express the siRNA-triggeringnucleic acid cassette and/or to express the RNA that hybridizes to theinitiation cleavage site in the initiator segment.

The siRNA-triggering nucleic acid cassettes can also be stacked with DNAimparting other traits of agronomic interest including DNA providingherbicide resistance or insect resistance such as using a gene fromBacillus thuringensis to provide resistance against lepidopteran,coliopteran, homopteran, hemiopteran, and other insects. Herbicides forwhich resistance is useful in a plant include glyphosate herbicides,phosphinothricin herbicides, oxynil herbicides, imidazolinoneherbicides, dinitroaniline herbicides, pyridine herbicides, sulfonylureaherbicides, bialaphos herbicides, sulfonamide herbicides and glufosinateherbicides. Persons of ordinary skill in the art are enabled inproviding stacked traits by reference to U.S. patent applicationpublications 2003/0106096A1 and 2002/0112260A1 and U.S. Pat. Nos.5,034,322; 5,776,760; 6,107,549 and 6,376,754 and toinsect/nematode/virus resistance by reference to U.S. Pat. Nos.5,250,515; 5,880,275; 6,506,599; 5,986,175 and U.S. Patent ApplicationPublication 2003/0150017 A1, all of which are incorporated herein byreference.

VIII. Constructs for Triggering RNAi

RNAi-inducing constructs contain an initiator (target) sequence and oneor more gene-suppressing elements in-phase or near-phase to theinitiation cleavage site in the in initiator (target) sequence. Theseare operably linked to a promoter or other regulatory sequence whichgoverns transcription from the RNAi-triggering cassette (comprising theinitiator sequence with an initiator cleavage site and at least one genesuppressing element upstream or downstream of the initiator sequence andthat may optionally overlap a portion of the initiator sequence) inorder to generate a single-stranded RNA comprising one or more elementsthat, when cleaved in register or nearly in register from the ininitiator cleavage site, yield one of more siRNA.

DNA constructs for plant transformation are assembled using methods wellknown to persons of ordinary skill in the art, and typically comprise apromoter operably linked to DNA, the expression of which provides anenhanced trait, e.g. by gene suppression using an siRNA-triggering (orRNAi-triggering) nucleic acid cassette alone or in combination with aDNA for expressing a protein or another RNA molecule. Other constructcomponents may include additional regulatory elements, such as 5′introns for enhancing transcription, 3′ untranslated regions (such aspolyadenylation signals and sites), DNA for transit or signal peptides.

Vectors suitable for stable transformation of culturable cells are wellknown. Typically, such vectors include a multiple-cloning site suitablefor inserting a cloned nucleic acid molecule, such that it will be underthe transcriptional control of 5′ and 3′ regulatory sequences. Inaddition, transformation vectors include one or more selectable markers;for bacterial transformation this is often an antibiotic resistancegene. A number of vectors suitable for stable transformation of plantcells or for the establishment of transgenic plants have been describedin, e.g., Pouwels et al. (Cloning Vectors: A Laboratory Manual, 1985,Suppl., 1987), Weissbach and Weissbach (Meth. Plant Mol. Bio., AcademicPress, 1989) and Gelvin et al. (Plant Molecular Biology Manual, KluwerAcademic Publishers, 1990). In addition, on of ordinary skill in the artis aware of the components useful in a transformation vector, and willbe able to select and assemble such components in order to tailor make avector for their specific use.

Typically, transformation and expression vectors include, for example,one or more cloned plant genes under the transcriptional control of 5′and 3′ regulatory sequences and a dominant selectable marker. Such plantexpression vectors also can contain a promoter regulatory region (e.g.,a regulatory region controlling inducible or constitutive,environmentally- or developmentally-regulated, or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

Also included in most expression vectors will be a promoter, which is anarray of nucleic acid control sequences which direct transcription of anucleic acid. A promoter includes necessary nucleic acid sequences nearthe start site of transcription, such as, in the case of an RNApolymerase II type promoter, a TATA element. Optionally, a promoter mayinclude an enhancer and/or a repressor element Enhancer and repressorelements can be located adjacent to, or distal to the promoter, and canbe located as much as several thousand base pairs from the start site oftranscription. Examples of promoters that can be used in the presentdisclosure include, but are not limited to the Cauliflower mosaic virus35S promoter, SV40 promoter, the CMV enhancer-promoter, the CMVenhancer/β-actin promoter, and the tissue-specific promoter probasin.Other promoter sequences that can be used to construct nucleic acids andpractice methods disclosed herein include, but are not limited to: thelac system, the trp system, the tac system, the trc system, majoroperator and promoter regions of phage lambda, the control region of fdcoat protein, the early and late promoters of SV40, promoters derivedfrom polyoma, adenovirus, retrovirus, baculovirus and simian virus, thepromoter for 3-phosphoglycerate kinase, the promoters of yeast acidphosphatase, the promoter of the yeast alpha-mating factors, anyretroviral LTR promoter such as the RSV promoter; inducible promoters,such as the MMTV promoter; the metallothionein promoter; heat shockpromoters; the albumin promoter; the histone promoter; the α-actinpromoter; TK promoters; B19 parvovirus promoters; the SV10 latepromoter; the ApoAI promoter and combinations thereof.

In certain embodiments, a promoter is a strong promoter, which promotestranscription of RNA at high levels, for example at levels such that thetranscriptional activity of the promoter generally accounts for about 5%or more of the transcriptional activity of all transcription within acell. The strength of a promoter is often tissue-specific and thus mayvary from one cell type to another. Examples of strong promotersinclude, but are not limited to: viral promoters (such as CaMV 35S orCoYMV), ubiquitin promoter (such as Ubi-1 from maize), actin promoter(e.g, Act from rice), nopaline synthase promoter, and the octopinesynthase promoter, pEMU promoter, MAS promoter, or a H3 histonepromoter.

In another embodiment, a promoter is a tissue-specific, cell-specific,or developmental stage-specific promoter, which promotes transcriptionin a single cell or tissue type, a narrow range of cells or tissues, orin one or more specific developmental stages, or at least promotesmeasurable more transcription in such. Examples of such promotersinclude, but are not limited to: anther-specific, embryo-specific,endosperm-specific, floral-specific, leaf-specific, meristem-specific,nodule-specific, phloem-specific, seed-specific, stem-specific,stomata-specific, trichome-specific, root-specific, tapetum-specific,and xylem-specific promoters. See, for instance, Carpenter et al., ThePlant Cell 4:557-571, 1992, Denis et al., Plant Physiol. 101:1295-13041993, Opperman et al., Science 263:221-223, 1993, Stockhause et al., ThePlant Cell 9:479-489, 1997; Roshal et al., EMBO J. 6:1155, 1987;Schernthaner et al., EMBO J. 7:1249, 1988; and Bustos et al., Plant Cell1:839, 1989.

Inducible promoters or gene-switches are used to both spatially andtemporally regulate gene expression. By allowing the time and/orlocation of gene expression to be precisely regulated, gene-switches orinducible promoters may control deleterious and/or abnormal effectscaused by overexpression or non-localized gene expression. Thus, for atypical inducible promoter in the absence of the inducer, there would belittle or no gene expression while, in the presence of the inducer,expression should be high (i.e., off/on). Examples ofstimulus-responsive promoters include, but are not limited tohormone-responsive promoters (e.g, ethanol inducible alcR-encodedtranscriptional activator (ALCR), a promoter derived from alcA),light-inducible promoters (such as a rbcS promoter), metal-induciblepromoters, heat-shock promoters, wound-inducible and stress-inducible(e.g., drought stress, salt stress, shear stress, nutrient stress)promoters. Others are activated by chemical stimuli, such as IPTG orTetracycline (Tet), or galactose. Other promoters are responsive topathogen infection or insect damage.

A number of controllable gene expression systems have been devised,including those regulated by light (e.g., the pea rbcS-3A promoter,Kuhlemeier et al., The Plant Cell, 1:471-478, 1989, and the maize rbcSpromoter, Schaffner and Sheen, Plant Cell 3:997, 1991), heat (Callis etal., Plant Physiol. 88:965, 1988; Ainley and Key, Plant Mol. Biol.,14:949-967, 1990; Holtorf et al., Plant Mol. Biol. 29:637-646, 1995),pathogens (PR1-a; Williams et al., Biotechnology 10:540-543, 1992; Gatz,Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:89-108, 1997), herbicidesafeners (In2-2, GST-27; De Veylder et al., Plant Cell Physiol.38:568-577, 1997), light (Kuhlemeier et al., Plant Cell 1:471-478,1989), wounding (Firek et al. Plant Mol. Biol. 22:129-212, 1993),ethanol (Salter et al., Plant J. 16:127-132, 1998), phytohormones (Li etal., Plant Cell 3:1167-1175, 1991), steroids (Aoyama and Chua, Plant J.,11:605-612, 1997), wounding (e.g., wunI, Siebertz et al., Plant Cell1:961, 1989), hormones, such as abscisic acid (Marcotte et al., PlantCell 1:969, 1989); chemicals such as methyl jasminate or salicylic acid(see Gatz et al., Ann. Rev. Plant Physiol. Plant Mol. Biol. 48:89-1081997), and tetracycline (Gatz et al., Plant J. 2:397-404, 1992; Weinmannet al., Plant J., 5:559-569, 1994; Sommer et al., Plant Cell Rep.17:891-896, 1998) (from Granger & Cyr, Plant Cell Reports 20:227-234,2001).

It is specifically contemplated that useful promoters will includepromoters present in plant genomes as well as promoters from othersources, including nopaline synthase (nos) promoter and octopinesynthase (ocs) promoters carried on tumor-inducing plasmids ofAgrobacterium tumefaciens, caulimovirus promoters such as thecauliflower mosaic virus or figwort mosaic virus promoters. Forinstance, see U.S. Pat. Nos. 5,322,938 and 5,858,742 which discloseversions of the constitutive promoter derived from cauliflower mosaicvirus (CaMV35S), U.S. Pat. No. 5,378,619 which discloses a FigwortMosaic Virus (FMV) 35S promoter, U.S. Pat. No. 5,420,034 which disclosesa napin promoter, U.S. Pat. No. 6,437,217 which discloses a maize RS81promoter, U.S. Pat. No. 5,641,876 which discloses a rice actin promoter,U.S. Pat. No. 6,426,446 which discloses a maize RS324 promoter, U.S.Pat. No. 6,429,362 which discloses a maize PR-1 promoter, U.S. Pat. No.6,232,526 which discloses a maize A3 promoter, U.S. Pat. No. 6,177,611which discloses constitutive maize promoters, U.S. Pat. No. 6,433,252which discloses a maize L3 oleosin promoter, U.S. Pat. No. 6,429,357which discloses a rice actin 2 promoter and intron, U.S. Pat. No.5,837,848 which discloses a root specific promoter, U.S. Pat. No.6,084,089 which discloses cold inducible promoters, U.S. Pat. No.6,294,714 which discloses light inducible promoters, U.S. Pat. No.6,140,078 which discloses salt inducible promoters, U.S. Pat. No.6,252,138 which discloses pathogen inducible promoters, U.S. Pat. No.6,175,060 which discloses phosphorus deficiency inducible promoters,U.S. Pat. No. 6,635,806 which discloses a coixin promoter, U.S.2002/0192813 A1 which discloses 5′, 3′ and intron elements useful in thedesign of effective plant expression vectors, U.S. 2004/0216189 A1 whichdiscloses a maize chloroplast aldolase promoter, and U.S. 2004/0123347A1 which discloses water-deficit inducible promoters, all of which areincorporated herein by reference. These and numerous other promotersthat function in plant cells are known to those skilled in the art andavailable for use in recombinant polynucleotides of the presentdisclosure to provide for expression of desired genes in transgenicplant cells.

Furthermore, the promoters may be altered to contain multiple “enhancersequences” to assist in elevating gene expression. Such enhancers areknown in the art. By including an enhancer sequence with suchconstructs, the expression of the selected protein may be enhanced.These enhancers often are found 5′ to the start of transcription in apromoter that functions in eukaryotic cells, but can often be insertedupstream (5′) or downstream (3′) to the coding sequence. In someinstances, these 5′ enhancing elements are introns. Particularly usefulas enhancers are the 5′ introns of the rice actin 1 (see U.S. Pat. No.5,641,876) and rice actin 2 genes, the maize alcohol dehydrogenase geneintron, the maize heat shock protein 70 gene intron (U.S. Pat. No.5,593,874) and the maize shrunken 1 gene.

In other aspects, sufficient expression in plant seed tissues is desiredto effect improvements in seed composition. Exemplary promoters for usefor seed composition modification include promoters from seed genes suchas napin (U.S. Pat. No. 5,420,034), maize L3 oleosin (U.S. Pat. No.6,433,252), zein Z27 (Russell et al. (1997) Transgenic Res.6(2):157-166), globulin 1 (Belanger et al (1991) Genetics 129:863-872),glutelin 1 (Russell (1997) supra), and peroxiredoxin antioxidant (Per1)(Stacy et al. (1996) Plant Mol. Biol. 31(6):1205-1216).

Recombinant DNA constructs prepared in accordance with this disclosurewill often include a 3′ element that typically contains apolyadenylation signal and site, especially if the recombinant DNA isintended for protein expression as well as gene suppression. Well-known3′ elements include those from Agrobacterium tumefaciens genes such asnos 3′, tml 3′, tmr 3′, tms 3′, ocs 3′, tr7 3′, e.g. disclosed in U.S.Pat. No. 6,090,627, incorporated herein by reference; 3′ elements fromplant genes such as wheat (Triticum aesevitum) heat shock protein 17(Hsp17 3′), a wheat ubiquitin gene, a wheat fructose-1,6-biphosphatasegene, a rice glutelin gene a rice lactate dehydrogenase gene and a ricebeta-tubulin gene, all of which are disclosed in U.S. published patentapplication 2002/0192813 A1, incorporated herein by reference; and thepea (Pisum sativum) ribulose biphosphate carboxylase gene (rbs 3′), and3′ elements from the genes within the host plant.

Constructs and vectors may also include a transit peptide for targetingof a gene target to a plant organelle, particularly to a chloroplast,leucoplast or other plastid organelle. For descriptions of the use ofchloroplast transit peptides see U.S. Pat. No. 5,188,642 and U.S. Pat.No. 5,728,925, incorporated herein by reference. For description of thetransit peptide region of an Arabidopsis EPSPS gene useful in theprovided constructs; see Klee et al (MGG 210:437-442, 1987).

For expression of constructs in fungi such as yeast, there are a varietyof promoters to choose from for various purposes. The following areprovided by way of example, and are not meant to be in any way limiting:

The Gal 1,10 promoter: This promoter is inducible by galactose. It canbe used to turn expression of an associated nucleic acid on and off, forinstance in order to follow the time dependent effects of expression.The Gal promoter is slightly leaky, and so is appropriate where it isnot essential to have absolutely no expression of the passenger gene inthe absence of galactose. The Gal 1 gene and Gal 10 gene are adjacentand transcribed in opposite directions from the same promoter region.The regulatory region containing the UAS sequences can be cut out on aDdeI Sau3A fragment and placed upstream of any other gene to confergalactose inducible expression and glucose repression.

PGK, GPD and ADH1 promoters: These are high expression constitutivepromoters. PGK=phosphoglycerate kinase, GPD=glyceraldehyde 3 phosphatedehydrogenase, ADH1=alcohol dehydrogenase

ADH2 promoter: This gene is glucose repressible and it is stronglytranscribed on non-fermentable carbon sources (similar to GAL 1,10,except not inducible by galactose).

CUP1 promoter: This is the metalothionein gene promoter. It is activatedby copper or silver ions added to the medium. The CUP1 gene is one of afew yeast genes that is present in yeast in more than one copy.Depending on the strain, there can be up to eight copies of this gene.By way of example, a gene, when placed under CUP1 regulation, should eprovided with a degree of control of the level of expression based onthe amount of copper (or silver) in the medium. Copper is toxic and anycells should be tested to see how well it tolerates copper before makinga CUP1 construct.

PHO5 promoter: This promoter is derived from a gene that encodes an acidphosphatase. It is induced by low or no phosphate in the medium. Thephosphatase is secreted in the chance it will be able to free up somephosphate from the surroundings. When phosphate is present, no PHO5message can be found. When phosphate is absent, the promoter is stronglyturned on.

Steroid inducible expression: Keith Yamamoto's lab has developed aninducible system in yeast similar to the ecdysone system for mammaliancells. The rat glucocorticoid receptor gene has been inserted behind theconstitutive GPD promoter to express the rat glucocorticoid receptor inyeast. A second vector was made with three glucocorticoid responseelements upstream of the CYC1 gene minimal promoter (cytochrome c gene).A cloning site was placed after this so a selected gene or otherengineered construct could be placed under control of the 3GRE/CYC1promoter. Both vectors are high copy vectors. This system works wellwith dose dependent expression, when steroid hormone is added to themedium. Response time is rapid with t_(1/2) of 7-9 minutes afteraddition of hormone.

Heat shock expression: By placing the UAS from a heat shock gene infront of the minimal CYC1 promoter, any gene or synthetic construct canbe placed under heat shock induction. This is a specialized requirementusually used in studies of heat shock response, or in regulation of RNAiunder different temperature regimens.

GAL1-10 promoter: This promoter is highly regulatable by galactose, suchthat there is a basal level on glucose, but over 100 fold increase whencells are placed in galactose medium.

The yeast GAL genes form one of the most intensely studied model systemsfor eukaryotic gene regulation. The structural genes, e.g. GAL1 andGAL10, are induced to very high level expression in galactose by theaction of the activator Gal4p. Gal4p binds to activation sequences(UASG) that lie up stream of GAL genes and activates transcription in aprocess that depends on gene-proximal TATA elements and involvesnumerous coactivators and general transcription factors including TBP.The activation function of Gal4p is modulated by Gal80p, an inhibitoryregulator that binds specifically to the activation domain of Gal4p,thus preventing gene activation in nongalactose carbon sources.

In certain embodiments, the provided constructs or methods are used orcarried out in animal cells, particularly cells from the nematode C.elegans. In such embodiments, promoters or other regulatory sequencesthat function in animal cells are useful. Myriad animal promoters arewell known to those of ordinary skill in the art, including constitutivepromoters and inducible or repressible promoters, as well as promotersthat show cell or tissue specificity or other regulated expression.Where a siRNA triggering cassette is expressed in C. elegans or a cellfrom a C. elegans organism, optionally a C. elegans promoter can beused. See, for instance published U.S. application Ser. No. 10/239,249(2003-0177507) and Ser. No. 09/422,569 (2003-0023997), which describethe use of various promoters for construct expression in theinvertebrate animal C. elegans. Specific examples of C. eleganspromoters include the following: unc-54, hsp16-2, unc-119, G_(0A1) andsel-12. It is also appropriate to use heterologous promoters in animalcells, including cells from (or in) C. elegans organisms. Additionalpromoters and/or regulatory sequences are discussed elsewhere in thisdocument.

Plant expression vectors optionally include RNA processing signals,e.g., introns, which may be positioned upstream or downstream of apolypeptide-encoding sequence in the transgene. In addition, theexpression vectors may also include additional regulatory sequences fromthe 3′-untranslated region of plant genes, e.g., a 3′ terminator regionto increase mRNA stability of the mRNA, such as the PI-II terminatorregion of potato or the octopine or nopaline synthase 3′ terminatorregions.

Such vectors also generally include one or more dominant selectablemarker genes, including genes encoding antibiotic resistance (e.g.,resistance to hygromycin, kanamycin, bleomycin, G418, streptomycin,paromomycin, or spectinomycin) and herbicide-resistance genes (e.g.,resistance to phosphinothricin acetyltransferase or glyphosate) tofacilitate manipulation in bacterial systems and to select fortransformed plant cells.

Screenable markers are also used for cell transformation, such as fungusor plant cell transformation, including color markers such as genesencoding β-glucuronidase (gus) or anthocyanin production, or fluorescentmarkers such as genes encoding luciferase or green fluorescence protein(GFP).

IX. In Vitro Production of Oligonucleotides

Though it is often appropriate to produce RNAi triggering constructsthrough genetic engineering techniques such as those discussed above, insome instances components of such constructs can be advantageouslyproduced using in vitro chemical synthesis.

In vitro methods for the synthesis of oligonucleotides are well known tothose of ordinary skill in the art; such conventional methods can beused to produce IROs for the disclosed methods. The most common methodfor in vitro oligonucleotide synthesis is the phosphoramidite method,formulated by Letsinger and further developed by Caruthers (Caruthers etal., Chemical synthesis of deoxyoligonucleotides, in Methods Enzymol.154:287-313, 1987). This is a non-aqueous, solid phase reaction carriedout in a stepwise manner, wherein a single nucleotide (or modifiednucleotide) is added to a growing oligonucleotide. The individualnucleotides are added in the form of reactive 3′-phosphoramiditederivatives. See also, Gait (Ed.), Oligonucleotide Synthesis. Apractical approach, IRL Press, 1984.

In general, the synthesis reactions proceed as follows: First, adimethoxytrityl or equivalent protecting group at the 5′ end of thegrowing oligonucleotide chain is removed by acid treatment. (The growingchain is anchored by its 3′ end to a solid support such as a siliconbead.) The newly liberated 5′ end of the oligonucleotide chain iscoupled to the 3′-phosphoramidite derivative of the next deoxynucleosideto be added to the chain, using the coupling agent tetrazole. Thecoupling reaction usually proceeds at an efficiency of approximately99%; any remaining unreacted 5′ ends are capped by acetylation so as toblock extension in subsequent couplings. Finally, the phosphite triestergroup produced by the coupling step is oxidized to the phosphotriester,yielding a chain that has been lengthened by one nucleotide residue.This process is repeated, adding one residue per cycle. See, forinstances, U.S. Pat. Nos. 4,415,732, 4,458,066, 4,500,707, 4,973,679,and 5,132,418. Oligonucleotide synthesizers that employ this or similarmethods are available commercially (e.g., the PolyPlex oligonucleotidesynthesizer from Gene Machines, San Carlos, Calif.). In addition, manycompanies will perform such synthesis (e.g., Sigma-Genosys, TX; OperonTechnologies, CA; Integrated DNA Technologies, IA; and TriLinkBioTechnologies, CA).

Oligonucleotides are conveniently available commercially up toapproximately 125 nucleotides; beyond this length the efficiency andpurification drops. Modified nucleotides can be incorporated into anoligonucleotide essentially as described above for non-modifiednucleotides.

Methods described above, or other methods known to those of ordinaryskill in the art, can be used to produce oligonucleotides comprising aninitiation sequence, a gene suppressing element, or combinationsthereof, for instance. Such oligonucleotides can be used to constructRNA-trigger nucleic acid cassettes, for instance.

X. Plants for Production of siRNAs

The presence of the cellular systems described herein necessary torespond to initiator sequences, and thereby produce siRNAs from thedescribed constructs, appears to be nearly universal within the plantand fungal kingdoms. These systems are also present in someinvertebrates, such as C. elegans. At the molecular level for instance,DCL and RDR homologs have been found in a variety of plant and fungispecies, as well as C. elegans. Thus, expression of target genes usingthe synthetic siRNA-bearing constructs (RNAi-triggering nucleic acidmolecules) described herein may be modified, particularly inhibited, ina wide range of target organisms and cells of such organisms. Theseinclude plants, including both monocotyledonous and dicotyledonousplants. The described system for inducing RNAi finds equal applicationin fungal systems, including filamentous (mold-type) and some yeast-typefungi, as well as C. elegans, a representative invertebrate animal.

Representative, non-limiting example plants include Arabidopsis; fieldcrops (e.g. alfalfa, barley, bean, clover, corn, cotton, flax, lentils,maize, pea, rape/canola, rice, rye, safflower, sorghum, soybean,sunflower, tobacco, and wheat); vegetable crops (e.g. asparagus, beet,brassica generally, broccoli, Brussels sprouts, cabbage, carrot,cauliflower, celery, cucumber (cucurbits), eggplant, lettuce, mustard,onion, pepper, potato, pumpkin, radish, spinach, squash, taro, tomato,and zucchini); fruit and nut crops (e.g. almond, apple, apricot, banana,blackberry, blueberry, cacao, cassava, cherry, citrus, coconut,cranberry, date, hazelnut, grape, grapefruit, guava, kiwi, lemon, lime,mango, melon, nectarine, orange, papaya, passion fruit, peach, peanut,pear, pineapple, pistachio, plum, raspberry, strawberry, tangerine,walnut, and watermelon); tree woods and ornamentals (e.g. alder, ash,aspen, azalea, birch, boxwood, camellia, carnation, chrysanthemum, elm,fir, ivy, jasmine, juniper, oak, palm, poplar, pine, redwood,rhododendron, rose and rubber).

XI. Delivery of Constructs to Target Cells

Once a nucleic acid molecule (e.g., synthetic construct) encoding atleast one siRNA for use in RNAi is generated, standard techniques may beused to express the encoded siRNA molecule(s) in transgenic plants,yeast, or animals. The basic approach is to clone, for instance, thesynthetic siRNA construct into a transformation vector, such that it isoperably linked to control sequences (e.g., a promoter) that directexpression of the nucleic acid in target cells. The transformationvector is then introduced into the target cells by one of a number oftechniques (e.g., electroporation) and progeny containing the introducednucleic acid construct are selected. In some embodiments, all or part ofthe transformation vector will stably integrate into the genome of thetarget cell. That part of the transformation vector that integrates intothe target cell and that contains the introduced synthetic siRNAconstruct and associated sequences for controlling expression (theintroduced “transgene”) may be referred to as the recombinant expressioncassette.

Selection of progeny, for instance, progeny plants, yeast, orinvertebrate cells, containing the introduced transgene may be basedupon the detection of an altered phenotype. Such a phenotype may resultdirectly from the synthetic construct cloned into the transformationvector or may be manifested as enhanced (or reduced) resistance to achemical agent (such as an antibiotic) as a result of the inclusion of aselectable marker gene incorporated into the transformation vector.

Examples of the modification of plant characteristics by transformationwith cloned cDNA sequences are replete in the technical and scientificliterature. Selected examples, which serve to illustrate the knowledgein this field of technology, include: U.S. Pat. No. 5,451,514; U.S. Pat.No. 5,750,385; U.S. Pat. No. 5,583,021; U.S. Pat. No. 5,589,615; U.S.Pat. No. 5,268,526; U.S. Pat. No. 5,741,684; U.S. Pat. No. 5,773,692; WO96/13582; published U.S. application Ser. No. 10/450,412 (2004-0139494),Ser. No. 09/850,846 (2002-0147168). These examples include descriptionsof transformation vector selection, transformation techniques and theassembly of constructs designed to express or over-express theintroduced nucleic acid.

In light of the foregoing and the provision herein of methods forproducing siRNA-producing synthetic constructs governed by describedinitiator sequences, one of ordinary skill in the art will be able tointroduce such nucleic acid constructs into plants, fungi, and animals(particularly invertebrates) in order to produce specimens exhibitingRNAi of one or more target genes.

XII. Plant Transformation, Regeneration, and Selection

Transformation and regeneration of both monocotyledonous anddicotyledonous plant cells is routine, and the most appropriatetransformation technique will be determined by the practitioner. Thechoice of method will vary with the type of plant to be transformed;those skilled in the art will recognize the suitability of particularmethods for given plant types. Suitable methods may include, but are notlimited to: electroporation of plant protoplasts; liposome-mediatedtransformation; polyethylene glycol (PEG) mediated transformation;transformation using viruses; micro-injection of plant cells;micro-projectile bombardment of plant cells; vacuum infiltration; andAgrobacterium tumefaciens (AT) mediated transformation. Typicalprocedures for transforming and regenerating plants are described in thepatent documents listed at the beginning of this section.

Following transformation and regeneration of plants with thetransformation vector, transformed plants may be selected using adominant selectable marker incorporated into the transformation vector.Typically, such a marker will confer antibiotic resistance on theseedlings of transformed plants, and selection of transformants can beaccomplished by exposing the seedlings to appropriate concentrations ofthe antibiotic.

After transformed plants are selected and grown to maturity, they can beassayed using the methods described herein, and other methodsappropriate to the synthetic construct of the transgene, to determinewhether the passenger siRNA(s) are being produced, and/or whether thetarget gene(s) are measurably inhibited by RNAi as a result of theintroduced transgene.

Numerous methods for transforming plant cells with recombinant DNA areknown in the art and may be used. Two commonly used methods for planttransformation are Agrobacterium-mediated transformation andmicroprojectile bombardment. Microprojectile bombardment methods areillustrated in U.S. Pat. No. 5,015,580 (soybean); U.S. Pat. No.5,550,318 (corn); U.S. Pat. No. 5,538,880 (corn); U.S. Pat. No.5,914,451 (soybean); U.S. Pat. No. 6,160,208 (corn); U.S. Pat. No.6,399,861 (corn) and U.S. Pat. No. 6,153,812 (wheat) andAgrobacterium-mediated transformation is described in U.S. Pat. No.5,159,135 (cotton); U.S. Pat. No. 5,824,877 (soybean); U.S. Pat. No.5,591,616 (corn); and U.S. Pat. No. 6,384,301 (soybean), all of whichare incorporated herein by reference. For Agrobacterium tumefaciensbased plant transformation system, additional elements present ontransformation constructs will include T-DNA left and right bordersequences to facilitate incorporation of the recombinant polynucleotideinto the plant genome.

In general it is useful to introduce recombinant DNA randomly, i.e. at anon-specific location, in the genome of a target plant line. In specialcases it may be useful to target recombinant DNA insertion in order toachieve site-specific integration, e.g. to replace an existing gene inthe genome, to use an existing promoter in the plant genome, or toinsert a recombinant polynucleotide at a predetermined site known to beactive for gene expression. Several site specific recombination systemsexist which are known to function implants include cre-lox as disclosedin U.S. Pat. No. 4,959,317 and FLP-FRT as disclosed in U.S. Pat. No.5,527,695, both incorporated herein by reference.

Transformation methods are preferably practiced in tissue culture onmedia and in a controlled environment. “Media” refers to the numerousnutrient mixtures that are used to grow cells in vitro, that is, outsideof the intact living organism. Recipient cell targets include, but arenot limited to, meristem cells, callus, immature embryos and gameticcells such as microspores, pollen, sperm and egg cells. It iscontemplated that any cell from which a fertile plant may be regeneratedis useful as a recipient cell. Callus may be initiated from tissuesources including, but not limited to, immature embryos, seedling apicalmeristems, microspores and the like. Cells capable of proliferating ascallus are also recipient cells for genetic transformation. Practicaltransformation methods and materials for making transgenic plants, e.g.various media and recipient target cells, transformation of immatureembryos and subsequent regeneration of fertile transgenic plants aredisclosed in U.S. Pat. Nos. 6,194,636 and 6,232,526, which areincorporated herein by reference.

The seeds of transgenic plants can be harvested from fertile transgenicplants and be used to grow progeny generations of transformed plantsincluding hybrid plants line for screening of plants having an enhancedagronomic trait. In addition to direct transformation of a plant with arecombinant DNA, transgenic plants can be prepared by crossing a firstplant having a recombinant DNA with a second plant lacking the DNA. Forexample, recombinant DNA can be introduced into first plant line that isamenable to transformation to produce a transgenic plant which can becrossed with a second plant line to introgress the recombinant DNA intothe second plant line. A transgenic plant with recombinant DNA providingan enhanced agronomic trait, e.g. enhanced yield, can be crossed withtransgenic plant line having other recombinant DNA that confers anothertrait, e.g. herbicide resistance or pest resistance, to produce progenyplants having recombinant DNA that confers both traits. Typically, insuch breeding for combining traits the transgenic plant donating theadditional trait is a male line and the transgenic plant carrying thebase traits is the female line. The progeny of this cross will segregatesuch that some of the plants will carry the DNA for both parental traitsand some will carry DNA for one parental trait; such plants can beidentified by markers associated with parental recombinant DNA Progenyplants carrying DNA for both parental traits can be crossed back intothe female parent line multiple times, e.g. usually 6 to 8 generations,to produce a progeny plant with substantially the same genotype as oneoriginal transgenic parental line but for the recombinant DNA of theother transgenic parental line

In the practice of transformation DNA is typically introduced into onlya small percentage of target cells in any one transformation experiment.Marker genes are used to provide an efficient system for identificationof those cells that are stably transformed by receiving and integratinga transgenic DNA construct into their genomes. Preferred marker genesprovide selective markers which confer resistance to a selective agent,such as an antibiotic or herbicide. Any of the herbicides to whichplants may be resistant are useful agents for selective markers.Potentially transformed cells are exposed to the selective agent. In thepopulation of surviving cells will be those cells where, generally, theresistance-conferring gene is integrated and expressed at sufficientlevels to permit cell survival. Cells may be tested further to confirmstable integration of the exogenous DNA. Commonly used selective markergenes include those conferring resistance to antibiotics such askanamycin and paromomycin (nptII), hygromycin B (aph IV) and gentamycin(aac3 and aacC4) or resistance to herbicides such as glufosinate (bar orpat) and glyphosate (aroA or EPSPS). Examples of such selectable areillustrated in U.S. Pat. Nos. 5,550,318; 5,633,435; 5,780,708 and6,118,047, all of which are incorporated herein by reference. Screenablemarkers which provide an ability to visually identify transformants canalso be employed, e.g., a gene expressing a colored or fluorescentprotein such as a luciferase or green fluorescent protein (GFP) or agene expressing a beta-glucuronidase or uidA gene (GUS) for whichvarious chromogenic substrates are known.

Cells that survive exposure to the selective agent, or cells that havebeen scored positive in a screening assay, may be cultured inregeneration media and allowed to mature into plants. Developingplantlets can be transferred to plant growth mix, and hardened off,e.g., in an environmentally controlled chamber at about 85% relativehumidity, 600 ppm CO₂, and 25-250 microeinsteins M⁻² s⁻¹ of light, priorto transfer to a greenhouse or growth chamber for maturation. Plants areregenerated from about 6 weeks to 10 months after a transformant isidentified, depending on the initial tissue. Plants may be pollinatedusing conventional plant breeding methods known to those of skill in theart and seed produced, e.g. self-pollination is commonly used withtransgenic corn. The regenerated transformed plant or its progeny seedor plants can be tested for expression of the recombinant DNA andscreened for the presence of enhanced agronomic trait.

XIII. Transgenic Plants and Seeds

Transgenic plant seed provided herein are grown to generate transgenicplants having an enhanced trait as compared to a control plant. Suchseed for plants with enhanced agronomic trait(s) is identified byscreening transformed plants, progeny, or progeny seed for the enhancedtrait(s). For efficiency, a screening program is beneficially used toevaluate multiple transgenic plants (events) comprising the recombinantDNA, e.g. multiple plants from 2 to 20 or more transgenic events.

Transgenic plants grown from transgenic seed provided herein demonstrateimproved agronomic traits that contribute to increased yield or othertrait that provides increased plant value, including, for example,improved seed quality. Of particular interest are plants having enhancedyield resulting from improved plant growth and development, stresstolerance, improved seed development, higher light response, improvedflower development, or improved carbon and/or nitrogen metabolism

Many transgenic events which survive to fertile transgenic plants thatproduce seeds and progeny plants will not exhibit an enhanced agronomictrait. Screening is necessary to identify the transgenic plant havingenhanced agronomic traits from populations of plants transformed asdescribed herein by evaluating the trait in a variety of assays todetect an enhanced agronomic trait. These assays also may take manyforms, including but not limited to, analyses to detect changes in thechemical composition, biomass, physiological properties, morphology ofthe plant.

XIV. Targets for RNAi

The target gene can be in any cell derived from or contained in anyorganism. The organism can be a plant, an animal, or fungus, asdescribed herein. The target gene may be a cellular gene (i.e., derivedfrom a cell, as opposed to a virus or other exogenous source), anendogenous gene (i.e., a cellular gene found in the genome), a transgene(i.e., a gene construct inserted at an ectopic site in the genome of thecell), or a gene from a pathogen or invasive entity which is capable ofinfecting or infesting an organism from which the cell is derived.Specific, non-limiting examples of target genes include genes encoding:structural or regulatory molecules; enzymes; toxins; transcriptionfactors; chromatin factors; metabolic factors: secreted factors; mRNAexpressed by pathogens; reproductive factors; pigments; pathogenresponse factors; environmental stress factors; allergens; and so forth.Also contemplated are target genes that are involved in reproduction,particularly male fertility in plants; genes that enhance vegetativegrowth. Targets also can be selected from non-coding regions of thegenome of the target organism.

In addition to endogenous gene and non-gene targets, it is contemplatedthat the RNAi-triggering constructs and methods described herein can beused to inhibit expression of pathogen or parasite genes, for instancegene sequences expressed bacterial, viral, other pathogen, animal pest,or plant pest (e.g., nematode) targets. By way of example, such geneinhibition in the context of an organism infected or infested with suchpathogenic target could be used to combat the pathogen. Treatment ofpathogens using such a system could be preventative, wherein theRNAi-triggering construct(s) are introduced before there is knowninfection or introduction of the pathogenic organism. In suchembodiments, the presence of the RNAi-triggering system is intended toprevent, reduce, or ameliorate a subsequent infection or contaminationwith the target pathogen or other microorganism. Alternatively, infectedor infested organisms could be treated after the microorganism(s) arepresent. In such embodiments, the RNAi-triggering system is intended totreat or eradicate the infection/infestation.

In yet other embodiments, an RNAi-triggering system is introduced toprovide inhibitory control over a transgenic target gene sequence, orset of transgenic sequences, for instance that have been introduced intoa transgenic plant, fungus, or other cell. Such targets might includetransgenes that confer desirable or undesirable traits to the targetorganism. Representative non-limiting examples of categories oftransgenes are discussed herein; any transgene could serve as a target,and specific targets will be best selected by the practitioner.

Inhibition of target gene expression or activity can be measured bymonitoring the levels of target gene mRNA or proteins encoded by thetarget gene. Examples of known techniques used to measure mRNA levelsinclude RNA solution hybridization, nuclease protection, Northern blotanalysis, and reverse transcription which can be used in combinationwith polymerase chain reaction. Examples of techniques used to measuretarget gene protein levels include antibody binding, enzyme linkedimmunosorbent assay (ELISA), Western blot analysis, immunoassays (e.g.radioimmunoassay), and fluorescence activated cell sort (FACS).

Depending on the particular target gene and the level of production ofthe siRNA, increasing the production of siRNA(s), for example throughexpression from a transgene described herein, may provide partial orcomplete loss of expression, or function, of the target gene. Theinhibition in target gene expression in different embodiments is atleast a 5%, at least a 10%, at least a 20%, at least a 30%, at least a50%, at least a 75%, at least an 80%, at least an 85%, at least a 90%,at least a 95%, or a 100% inhibition in target gene expression.

XV. Regulated RNAi

The RNAi-triggering systems described herein can further be employed toexploit differentially regulated systems within a target, for instancein order to provide cell-specific, tissue-specific, or developmentallyspecific RNAi of one or more specific genes. In particular, miRNAsfrequently accumulate in specific cell-types or tissues (e.g. Palatniket al., Nature 425:257-263, 2003) or are induced under specificconditions, such as nutrient or abiotic stress (Jones-Rhoades & Bartel,Mol Cell 14:787-799, 2004). Thus, cell-, tissue-, or conditional RNAimay be regulated by cell-, tissue- or condition-specific miRNA or siRNAexpression by employing a target sequence (initiator sequence) thatinteracts with a specific regulated small RNA to guide cleavage of thetarget sequence in the desired expression pattern. Representative miRNAsand functions associated with their target(s) are listed in Table 4.

Alternatively, or in combination, regulated RNAi can also be achievedusing expression cassettes that are only transcribed, or preferentiallytranscribed, in certain cells, tissues, conditions, and so forth.Represented promoters useful for such regulated expression are discussedherein.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the invention to the particular features or embodiments described.

Example 1 MiRNA-Directed Phasing During Trans-Acting siRNA Biogenesis inPlants Small RNA Blot Analysis

Low molecular weight RNA (5 μg) from Arabidopsis inflorescence tissuewas used for miRNA and endogenous siRNA analysis as described (Allen etal., Nat Genet. 36:1282-1290, 2004). Mutant lines for dcl1-7, dcl2-1,dcl3-1, rdr1-1, rdr2-1, hen1-1, hyl1-2, rdr6-11, rdr6-15, and sgs3-11were described previously (Allen et al., Nat Genet. 36:1282-1290, 2004;Park et al., Curr Biol 12:1484-1495, 2002; Peragine et al., Genes & Dev18:2369-2379, 2004; Vazquez et al., Curr Biol 14:346-351, 2004a; Xie etal., PLoS Biol 2:642-652, 2004). The hst-15 allele used was theSALK_(—)079290 T-DNA insertion line from ABRC, which contains a T-DNA atposition 1584 from the start codon. Probes for miR159, miR167, andAtSN1-siRNA blots were described previously (Llave et al., Plant Cell14:1605-1619, 2000a; Zilberman et al., Science 299:716-719, 2003). Allother miRNAs were detected using end-labeled DNA oligonucleotides.Probes for ta-siRNA loci were PCR amplified from Col-0 genomic DNA,cloned into pGEMT-Easy, and verified by sequencing. Radiolabeled probesincorporating ³²P-UTP were made by T7 RNA polymerase transcription, toobtain strand specific small RNA probes. Probes were as follows: TAS3locus, Chr3:5862146-5862295; At3g39680 (TAS2) locus,Chr2:16546831-16547300.

Computational Prediction of miRNA Targets

An initial pool of predicted target sites for validated miRNAs wascreated by FASTA searches using a +15/−10 scoring matrix of the TAIR AGItranscript database, limited to 4 mispairs, 4G:U pairs, to a total ofseven, with 100,000 results obtained for the reverse complement of eachsmall RNA. A single, one nucleotide gap was allowed. The miRNA targetprediction algorithm used to score these sites was developed based on 94experimentally validated and predicted family members of miRNA-targetsite duplexes, including 55 targets validated in previous studies, 11new validated targets, plus 28 family members with conserved miRNAtarget sites (Target Rule Set, Table 3). Three filters based on theTarget Rule Set were applied sequentially. In each case, base one isconsidered to be the first nucleotide from the 5′ end of the miRNA.First, targets with a mismatch score greater than four were excluded.The Minimum Free Energy (ΔG_(MFE)) of a perfect miRNA-target duplex wasdetermined by computationally attaching a perfectly complementary targetsequence to a small RNA using a four base “gap” linker sequence (----).The free energy each miRNA-predicted target site (ΔG_(target)) wasdetermined by computationally linking the target sequence to the smallRNA, from which the MFE ratio was calculated (ΔG_(target)/ΔG_(MFE)). Allthermodynamic values were calculated using RNAFold in the Vienna RNApackage. Remaining targets with an MFE ratio less than 0.73 wereexcluded. Conservation of the target sequence was determined by usingthe region containing the target sequence in a BLAST search against theArabidopsis transcript and EST databases, NCBI EST database, and O.sativa Unigene database, and removing any targets with no matches withless than three base changes in the target sequence. Duplicate targetsites (identical genes) for related miRNA family members were combinedin the final target gene set.

TABLE 3 Summary of miRNA target gene predictions represented in FIG. 1Rule Original miRNA Development prediction Systematic name^(a) Commonname^(a) Gene family family Set Score^(b) MFE Ratio Pass/Fail referenceBin 1. Previously predicted miRNA target genes, experimentally validated 1 At1g27370 SPL10 SPL miR156 yes 3 0.808 Pass c  2 At5g43270 SPL2 SPLmiR156 yes 3 0.842 Pass c  3 At1g53160 SPL4 SPL miR157 yes 3 0.820 Passc  4 At5g06100 MYB33 MYB miR159 yes 3 0.787 Pass c; d  5 At3g11440 MYB65MYB miR159 yes 3 0.787 Pass c; d  6 At1g77850 ARF17 ARF miR160 yes 0.50.990 Pass c  7 At2g28350 ARF10 ARF miR160 yes 2 0.844 Pass c  8At4g30080 ARF16 ARF miR160 yes 2.5 0.863 Pass c  9 At1g06580 PPRmiR161.1 yes 3 0.713 Fail c 10 At1g63150 PPR miR161.2 yes 1.5 0.856 Passc 11 At5g41170 PPR miR161.1 yes 1 0.792 Pass c 12 At1g1040 DCL1 DCLmiR162 yes 2 1.000 Pass e 13 At1g66690 SAMT miR163 yes 1 0.898 Pass d 14At1g66700 SAMT miR163 yes 1 0.898 Pass d 15 At1g66720 SAMT miR163 yes 20.886 Pass f 16 At3g44860 SAMT miR163 yes 3 0.765 Pass f 17 At1g56010NAC1 NAC miR164 yes 2 0.823 Pass c 18 At3g15170 CUC1 NAC miR164 yes 30.856 Pass c 19 At5g07680 NAC miR164 yes 2 0.849 Pass c 20 At5g53950CUC2 NAC miR164 yes 3 0.856 Pass c 21 At5g61430 NAC miR164 yes 2 0.849Pass c 22 At1g30490 PHV HD-ZipIII miR166 yes 3 0.860 Pass c 23 At1g52150AtHB15 HD-ZipIII miR166 yes 2.5 0.867 Pass c 24 At2g34710 PHB HD-ZipIIImiR166 yes 3 0.860 Pass c 25 At5g60690 REV/IFL1 HD-ZipIII miR166 yes 30.860 Pass c 26 At1g30330 ARF6 ARF miR167 yes 3.5 0.844 Pass c; d 27At5g37020 ARF8 ARF miR167 yes 4 0.779 Pass c; d 28 At1g48410 AGO1 AGOmiR168 yes 4 0.735 Pass c 29 At1g17590 HAP2 miR169 yes 2.5 0.866 Pass c30 At1g54160 HAP2 miR169 yes 3 0.840 Pass c 31 At1g72830 HAP2c HAP2miR169 yes 2.5 0.834 Pass b 32 At3g05690 HAP2b HAP2 miR169 yes 3 0.746Pass b 33 At3g20910 HAP2 miR169 yes 4 0.735 Pass b 34 At5g06510 HAP2miR169 yes 3 0.746 Pass b 35 At2g45160 SCL6(II) SCL miR171 yes 0 1.000Pass g; c 36 At3g60630 SCL6(III) SCL miR171 yes 0 1.000 Pass g; c 37At4g00150 SCL6(IV) SCL miR171 yes 0 1.000 Pass g; c 38 At2g28550TOE1/RAP2.7 AP2 miR172 yes 3.5 0.857 Pass d 39 At4g36920 AP2 AP2 miR172yes 2.5 0.896 Pass d 40 At5g60120 TOE2 AP2 miR172 yes 1.5 0.928 Pass d41 At5g67180 TOE3 AP2 miR172 yes 3.5 0.896 Pass d 42 At1g30210 TCP24 TCPmiR319 yes 3.5 0.792 Pass i 43 At1g53230 TCP3 TCP miR319 yes 4 0.751Pass i 44 At2g31070 TCP10 TCP miR319 yes 3.5 0.777 Pass i 45 At3g15030TCP4 TCP miR319 yes 3.5 0.777 Pass i 46 At4g18390 TCP2 TCP miR319 yes3.5 0.792 Pass i 47 At1g12820 TIR/F-box miR393 yes 2 0.862 Pass b 48At3g23690 bHLH077 bHLH miR393 yes 3 0.871 Pass b 49 At3g26810 TIR/F-boxmiR393 yes 2 0.862 Pass b 50 At3g62980 TIR1 TIR/F-box miR393 yes 2.50.876 Pass b 51 At4g03190 TIR/F-box miR393 yes 3.5 0.761 Pass b 52At1g27340 F-box miR394 yes 1 0.820 Pass b 53 At5g43780 APS4 ATPsulfurylase miR395 yes 2 0.792 Pass b 54 At3g22890 APS1 ATP sulfurylasemiR395 yes 3.5 0.744 Pass b 55 At2g22840 GRF1 GRF miR396 yes 3.5 0.861Pass b 56 At2g36400 GRF3 GRF miR396 yes 3 0.861 Pass b 57 At2g45480 GRF9GRF miR396 yes 4 0.861 Pass b 58 At4g24150 GRF8 GRF miR396 yes 3.5 0.861Pass b 59 At4g37740 GRF2 GRF miR396 yes 3.5 0.861 Pass b 60 At5g53660GRF7 GRF miR396 yes 3.5 0.861 Pass b 61 At2g29130 Laccase miR397 yes 3.50.755 Pass b 62 At2g38080 Laccase miR397 yes 2.5 0.877 Pass b 63At5g60020 Laccase miR397 yes 2.5 0.828 Pass b 64 At3g15640 Cytochrome Coxidase miR398 yes 3 0.804 Pass b 65 At1g08830 CSD1 Copper superoxidedismutase miR398 yes 5 0.712 Fail b 66 At2g28190 CSD2 Copper superoxidedismutase miR398 yes 6.5 0.761 Fail b Bin 2. Previously predicted miRNAtarget gene, computational prediction only^(c)  1 At1g27360 SPL11 SPLmiR156 yes 3 0.808 Pass c  2 At1g69170 SPL6 SPL miR156 yes 3 0.808 Passc  3 At2g33810 SPL3 SPL miR156 yes 3 0.808 Pass c  4 At2g42200 SPL9 SPLmiR156 yes 2 0.832 Pass c  5 At3g57920 SPL15 SPL miR156 yes 2 0.832 Passc  6 At5g50570 SPL13 SPL miR156 yes 2 0.832 Pass c  7 At5g50670 SPLmiR156 yes 2 0.832 Pass c  8 At3g15270 SPL5 SPL miR157 yes 4 0.778 Passc  9 At2g26950 MYB104 MYB miR159 yes 4 0.880 Pass c; d 10 At2g32460MYB101 MYB miR159 yes 3.5 0.802 Pass c 11 At3g60460 MYB125 MYB miR159yes 3.5 0.786 Pass c 12 At5g55020 MYB120 MYB miR159 yes 3.5 0.732 Passc; d 13 At2g26960 MYB81 MYB miR159 yes 4.5 0.719 Fail c 14 At4g26930MYB97 MYB miR159 yes 4 0.729 Fail c 15 At1g62670 PPR miR161.1 yes 30.765 Pass c 16 At1g64580 PPR miR161.1 yes 3.5 0.787 Pass c 17 At1g62720PPR miR161.1 yes 5 0.754 Fail c 18 At1g63080 PPR miR161.2 yes 4 0.732Pass c 19 At1g63400 PPR miR161.2 yes 2 0.846 Pass c 20 At5g16640 PPRmiR161.2 yes 2.5 0.715 Fail c 21 At3g44870 SAMT miR163 yes 3 0.765 Passf 22 At5g39610 NAC miR164 yes 3.5 0.763 Pass b 23 At4g32880 AtHB8 HD-ZipmiR166 yes 3 0.860 Pass c 24 At5g12840 HAP2a HAP2 miR169 yes 3 0.735Pass b 25 At2g39250 SNZ AP2 miR172 yes 2.5 0.922 Pass h 26 At3g54990 SMZAP2 miR172 yes 1.5 0.954 Pass h 27 At4g14680 APS3 ATP sulfurylase miR395yes 3.5 0.744 Pass b 28 At3g52910 GRF4 GRF miR396 yes 3 0.861 Pass bAt3g28460 unclassified miR173 7 0.760 Fail, d not conserved At2g40760Rhodenase-like miR396 5.5 0.700 Fail, b not conserved At4g27180 ATK2Kinesin-like protein B miR396 6.5 0.527 Fail, b not conserved At5g12250Beta-6 tubulin miR397 10 0.698 Fail, b not conserved At3g54700 phosphatetransporter miR399 3.5 0.743 Fail, b not conserved Bin 3. New predictedmiRNA target genes from existing target families miRNA Systematicname^(a) Common name^(a) Gene family family Score^(b) MFE RatioPass/Fail 1 At1g62860 PPR miR161.1 4 0.749 Pass 2 At1g63330 PPR miR161.21 0.852 Pass 3 At1g62590 PPR miR161.2 1 0.852 Pass 4 At1g63630 PPRmiR161.2 2.5 0.859 Pass 5 At1g62930 PPR miR161.2 3 0.882 Pass 6At1g63130 PPR miR161.2 3 0.882 Pass 7 At1g62910 PPR miR161.2 3 0.882Pass 8 At1g63230 PPR miR161.2 3 0.735 Pass 9 At3g14020 HAP2 miR169 20.859 Pass Bin 4. Novel miRNA target genes, experimentally validatedmiRNA Systematic name^(a) Common name^(a) Gene family family Score^(b)MFE Ratio Pass/Fail Associated ESTs 1 At5g60760 2PGK miR447 3.5 0.807Pass 2 At5g10180 AST68 Sulfate transporter miR395 3 0.760 Pass 3At2g27400 TAS1a miR173 2.5 0.768 Pass CD534192, CD534180 4 At1g50055TAS1b miR173 4.5 Fail 5 At2g39675 TAS1c miR173 2.5 0.768 Pass 6At2g39681 TAS2 miR173 2.5 0.768 Pass BE521498 7 At3g17185 TAS3 miR3903.5 0.755 Pass AV534298, AI998599, BX838290, AA651246 8 At2g33770 E2-UBCmiR399 3.5 0.763 Pass 8 At1g31280 AGO2 AGO miR403 1 0.948 Pass BP648434,AU230620 Bin 5. Predicted miRNA target genes tested experimentally butnot validated miRNA Systematic name^(a) Common name^(a) Gene familyfamily Score MFE Ratio Pass/Fail Original prediction reference 1At1g64100 PPR miR158 4 0.733 Pass C 2 At3g03580 PPR miR158 3.5 0.770Pass 3 At2g03210 FUT2 FUT miR158 4 0.731 Pass 4 At2g03220 FUT1 FUTmiR158 4 0.737 Pass ^(a)Systematic and common names for genes were fromTAIR (available on the World Wide Web at arabidopsis.org) and AGRIS(available on-line at arabidopsis.med.ohio-state.edu/AtTFDB/index.jsp);^(b)Score was derived from a modified version of the scoring systemdeveloped by Jones-Rhoades et al., 2004. References: b: Jones-Rhoades etal., 2004; c: Rhoades et al., 2002; d: Park et al., 2002; e: Xie et al.,2003; f: Allen et al., 2004; g: Llave et al., 2002; h: Schmid et al.,2003; i:

Targets for ARF3 and ARF4 were predicted by aligning nucleotide sequencefrom orthologs from 17 selected species using TCoffee. Similarity over a21 nucleotide window (characteristic of a miRNA target site) was plottedusing PLOTCON in the EMBOSS software suite. Regions beyond the twopredicted target sites showing low nucleotide conservation were removedfor clarity. Orthologs of the At3g17185 were identified using BLAST,with ESTs only in the predicted miRNA orientation chosen. All selectedESTs were analyzed for the presence of an ARF gene or other conservedORF by BLASTX analysis against an Arabidopsis protein database, and anymatch eliminated. ESTs were aligned using TCoffee, and the poorlyconserved region surrounding the putative miRNAs removed.

Microarray Analysis

Inflorescence tissue (stages 1-12) was collected in triplicate, withthree bulked plants for each genotype per replicate. Controls for dcl1-1and hen1-1 were La-er, controls for hyl1-2, hst-15, dcl2-1, dcl3-1,rdr1-1, rdr2-1, and rdr6-15 were Col-0. RNA was extracted using Trizol,followed by purification using the Plant RNeasy Midi kit (Qiagen).Biotinylated cRNA was synthesized from 5 μg total RNA using theMessageAmp kit (Ambion). Twenty micrograms (20 μg) ofconcentration-adjusted cRNA were fragmented and hybridized to ATH1GeneChip arrays according to the manufacturer's protocol (Affymetrix).Samples were normalized using RMA Express (Bolstad et al.,Bioinformatics 19, 185-193, 2003), and imported into Genespring v7(Silicon Genetics) for analysis. Hierarchical clustering was performedusing the standard clustering algorithm.

5′ RACE Analysis of miRNA Directed Cleavage of Target Genes

Cleavage sites of miRNA target genes were mapped using the InvitrogenGeneRacer 5′ RACE procedure as described previously (Kasschau et al.,Dev Cell 4:205-217, 2003; Llave et al., Science 297:2053-2056, 2002).Gene specific primers were designed approximately 500 nucleotidesdownstream of the predicted cleavage site. These primers were used incombination with an adapter specific primer to amplify cleavage productsby PCR. Purified PCR products were cloned into pGEM-T Easy.

Phylogeny Reconstruction Methods

The phylogenetic tree for the ARF family was generated by aligning theconserved ARF domain using TCoffee, followed by Bayesian reconstructionof a consensus family tree (Allen et al., Nat Genet. 36:1282-1290,2004).

Results

Computational Prediction and Validation of New miRNA Targets

A rigorous set of computationally predicted and validated targets formost Arabidopsis miRNA families has emerged (Table 4 and Table 3)(Aukerman & Sakai, Plant Cell 15:2730-2741, 2003; Chen, Science303:2022-2025, 2004; Emery et al., Curr Biol 13:1768-1774, 2003;Jones-Rhoades & Bartel, Mol Cell 14:787-799, 2004; Kasschau et al., DevCell 4:205-217, 2003; Llave et al., Science 297:2053-2056, 2002b;Mallory et al., Curr Biol 14:1035-1046, 2004; Palatnik et al., Nature425:257-263, 2003; Park et al., Curr Biol 12:1484-1495, 2002; Rhoades etal., Cell 110:513-520, 2002; Tang et al., Genes & Dev 17:49-63 2003;Vaucheret et al., Genes Dev 18:1187-1197, 2004; Vazquez et al., CurrBiol 14:346-351, 2004a; Xie et al., Curr Biol 13:784-789, 2003).However, clear targets for several miRNAs (miR158, miR173, miR390/391,miR399, miR403 and miR447) are not yet known.

TABLE 4 Arabidopsis microRNA and ta-siRNA Target Families Small RNATarget Number of family^(a) family targets Target Function microRNA 1miR156^(b) SBP 11 transcription factor 2 miR158 3 miR159^(b) MYB 8transcription factor miR319^(b) TCP^(g) 5 transcription factor 4mir160^(b) ARF 3 transcription factor 5 miR161^(b) PPR 17 unknown 6miR162^(b) DCL 1 miRNA metabolism 7 miR163^(b) SAMT 5 metabolism 8miR164^(b) NAC 6 transcription factor 9 miR166^(b) HD-ZIPIII 5transcription factor 10 miR167^(b) ARF 2 transcription factor 11miR168^(b) AGO1 1 miRNA metabolism 12 miR169^(b) HAP2 8 transcriptionfactor 13 miR171^(b) SCR 3 transcription factor 14 miR172^(b) AP2 6transcription factor 15 miR173 TAS1, TAS2 4 ta-siRNA biogenesis 16miR390 TAS3 1 ta-siRNA biogenesis 17 miR393^(b) TIR1/F-box 4 hormonesignaling bHLH 1 transcription factor 18 miR394^(b) F-box 1 hormonesignaling 19 miR395^(b) ATPS 4 metabolism AST metabolism 20 miR396^(b)GRF 7 transcription factor 21 miR397^(b) laccase/Cu 3 metabolism oxidase22 miR398^(b) CSD 2 stress response CytC oxidase 1 metabolism 23 miR399E2-UBC 1 ubiquitin conjugation 24 miR447 2PGK 1 metabolism 25 miR403AGO2 1 miRNA metabolism 26 miR408 laccase 1 metabolism Trans-actingsiRNA 1 TAS1 unclassified^(s, t) 5 unknown 2 TAS2 PPR^(c) 8 unknown 3TAS3 ARF^(c) 4 transcription factor ^(a)miRNA families contain at leastone member, with related miRNAs with up to five changes grouped into afamily; ^(b)miRNAs with targets used in the Rule development set;^(c)targets families validated in previous studies are in blue, italicsindicated additional family members validated in this study, redindicates gene families validated only in this study.

To further extend and refine the analysis of miRNA targets in plants, wedeveloped a set of computational “rules” for Arabidopsis miRNA-targetinteractions involving 22 miRNA families. These were used to produce atarget prediction set that was experimentally tested (FIG. 1A). The ruledevelopment set included 66 experimentally validated targets and 28previously predicted targets that are closely related to validatedfamily members. Among the 66 validated targets were 55 previouslypublished targets and 11 new validated targets.

Experimental validation of targets involved 5′RACE assays to detect acleavage site opposite of position 10 from the 5′ end of the miRNA(Kasschau et al., Dev Cell 4:205-217, 2003; Llave et al., Science297:2053-2056, 2002). Detection of a cleavage product with a 5′ terminuscorresponding to the predicted miRNA-guided cleavage site is strongevidence in support of target site function. Validated targets includedgenes from multigene families in which closely related paralogs wereshown previously to be miRNA targets (Bins 1 and 3, FIG. 2A), and ninenovel targets discussed in detail below (Bin 4, FIGS. 3A and 3B).

Two parameters were analyzed for rule development. First, the occurrenceof mispaired bases between miRNAs and targets was analyzed. AllmiRNA-target duplexes within the rule set contained four or fewerunpaired bases, four or fewer G:U pairs, up to one single-nucleotidebulge, and a total of seven or fewer unpaired plus G:U positions. Thepositions of mispairs were examined by plotting the percentage ofmismatched and G:U pairs at each target nucleotide positions (countingfrom the 3′ end) (FIG. 1B). Nucleotide pairs at positions 2-13 formed acore segment with relatively few mismatches relative to positions 1 and14-21. This core segment is longer than the core segment of animalmiRNA-target duplexes (positions 2-8) (Lewis et al., Cell 115:787-798,2003). A mispair scoring system, modified from that used byJones-Rhoades and Bartel (Mol Cell 14:787-799, 2004), was applied toaccount for the reduced occurrence of mispairs within the core segment.Mismatched pairs or single nucleotide bulges were each scored as 1 andG:U pairs were scored as 0.5. Mismatches and G:U pair scores weredoubled within the core segment. A score of ≦4 captured 91 of 94 targetsin the rule development set for a false negative rate of 0.03.

Second, a relative thermodynamic parameter was investigated. The minimumfree energy (MFE) of a hypothetical duplex containing each of the 94targets paired with a perfectly complementary sequence (ΔG_(MFE)) wascalculated and compared to the free energy calculated for the actualmiRNA-target duplex (ΔG_(target)). The MFE ratio (ΔG_(target)/ΔG_(MFE))was calculated for each duplex in the rule set. Eighty-nine of theduplexes in the rule set had an MFE ratio ≧0.73 (FIG. 1C), correspondingto a false negative rate of 0.05. Combining the mispair (≦4) and MFEratio (≧0.73) limits in a series of filters resulted in capture of 87targets from the rule set (false negative rate=0.07). The mispair andMFE ratio limits were applied in searches using all validated miRNAsfrom the 25 families (Table 4) and the Arabidopsis transcript database,resulting in 145 prospective targets (FIG. 1D).

Target sequence conservation across species and between closely relatedparalogs was applied as a final filter. For all miRNAs that wereconserved between monocots and dicots (or between dicot families),predicted target sites were required to be similarly conserved(Jones-Rhoades & Bartel, Mol Cell 14:787-799, 2004). For non-conservedmiRNAs, targets sites were required to be present within more than oneparalog in Arabidopsis. When applied to the rule development set, therespective conservation filters resulted in loss of no genes.Application of the conservation filter to the 145 genes that passed themispair and MFE ratio filters resulted in 103 genes (FIG. 1A).

To further extend the chances for target identification, an miRNA targetsearch was also done using the Arabidopsis EST database. The samemispair and conservation filters were used, but the MFE ratio filterlimit was lowered to 0.70 to account for known sequencing errors withinthe EST dataset. A redundancy filter was added to subtract allprospective target genes that also passed the target search using thetranscript database. Six new prospective targets were identified in theEST search, resulting in a total of 109 predicted targets. These wereassigned to several bins (FIG. 1A, Table 3). Bin 1 contained 63 of 66previously validated targets that contributed to the rule set. Bin 2contained 24 of the 28 predicted targets from the rule set. Thus, theoverall false negative rate was 0.07. Bin 3 contained nine new predictedtargets from existing target gene families. These previouslynonpredicted targets included eight pentatricopeptide repeat (PPR) genestargeted by miR161.1 and miR161.2, a HAP2a gene (At1g14020) targeted bymiR169, and a sulfate transporter (AST68, At5g10180) gene targeted bymiR395. Bin 4 contained nine novel targets that were experimentallyvalidated and analyzed in detail (see following sections). Bin 5contained four genes that were predicted to interact with miR158, buteach of these failed the 5′RACE validation assay. If it is assumed thatBin 5 genes represent all incorrect predictions from this search, thenthe false positive rate was 0.04.

Genes encoding an E2-ubiquitin conjugating enzyme (E2-UBC, At2g33770),Argonaute2 (AGO2, At1g31280), and a 2-phosphoglycerate kinase (2PGK,At5g60760) were validated as targets of miR399, miR403 and miR477,respectively, and represent the only conventional genes in Bin 4 (FIG.3A). Possibly because of computational searches using a transcriptdatabase containing a misannotated E2-UBC, miR399 was predictedpreviously to target a different mRNA encoding a phosphate transporter(At3g54700) (Jones-Rhoades & Bartel, Mol Cell 14:787-799, 2004). Thisgene was not predicted in our analysis, and the 5′RACE assay failed toreveal a miR399-guided cleavage product. The E2-UBC target, which wasidentified here and predicted by Sunkar and Zhu (Plant Cell16:2001-2019, 2004) only using EST databases, contains up to fivemiR399-interacting sites in the 5′ untranslated region (UTR). Cleavageproducts were detected with 5′ termini corresponding to cleavage at fourof these sites, most prominently sites 2 and 3 (FIG. 3A). OrthologousE2-UBC genes in rice and at least three other plant species each contain3-5 conserved target sites. This is the only example of both a 5′UTRtarget position and multiple miRNA-target sites in plant genes. ThemiR403-target site was identified within the 3′UTR of the AGO2transcript from Arabidopsis and several other dicot families, but not inorthologous AGO2 transcripts from monocots. This is the secondmiRNA-targeted AGO family member identified, as AGO1 was shown to betargeted by miR168. Whereas AGO1 is required for miRNA activity(Vaucheret et al., Genes Dev 18:1187-1197, 2004), presumably withinRISC, a function for AGO2 is currently not known. The 2PGK gene(At5g60760) was validated as an miR447 target (FIG. 3A), and joins agrowing list of plant miRNA targets that encode proteins with metabolicfunctions (Jones-Rhoades & Bartel, Mol Cell 14:787-799, 2004).

The five remaining Bin 4 genes were validated as miR173 and miR390targets (FIG. 3B), and were predicted only from EST database due totheir unusual nature. These are discussed in detail below.

Expression Profiling of Predicted miRNA Targets

Most miRNAs of plants direct cleavage of their targets. Loss-of-functionmutations in miRNA metabolic or biogenesis genes, therefore, frequentlyresult in elevated target transcript levels (Kasschau et al., Dev Cell4:205-217, 2003; Palatnik et al., Nature 425:257-263, 2003; Vazquez etal., Curr Biol 14:346-351, 2004a; Xie et al., Curr Biol 13:784-789,2003). To systematically analyze the effects of miRNA and endogenoussiRNA defects on validated and predicted miRNA target genes inArabidopsis, expression profiling was done using nine mutant (condition)plants and two control plants. The mutants included miRNA-defectivedcl1-7, hen1-1 and hyl1-2 (Park et al., Curr Biol 12:1484-1495, 2002;Schauer et al., Trends Plant Sci 7:487-491, 2002; Vazquez et al., CurrBiol 14:346-351, 2004a), which were shown to reduce or eliminateaccumulation of miRNAs. A new insertion mutant, hst-15, with predicteddefects in nucleocytoplasmic transport of miRNA and ta-siRNA precursors(Bollman et al., Development 130:1493-1504, 2003) was used. Usinginflorescence tissue, hst-15 had only modest or no effects on miRNAaccumulation. However, as shown using the hst-1 mutant (Bollman et al.,Development 130:1493-1504, 2003; Peragine et al., Genes & Dev18:2369-2379, 2004), hst-15 had several developmental abnormalities,including a more rapid juvenile to adult phase change, leaf curling andepinasty, altered silique phyllotaxy and small flowers (FIG. 4A). Thehst-15 transcript accumulated to low levels specifically in the hst-15mutant plant; this was in contrast to the dcl1-7 transcript, which wasupregulated in each of the miRNA-defective mutants due to loss ofmiR162-mediated feedback regulation (Xie et al., Curr Biol 13:784-789,2003).

The mutant series also included five siRNA-defective mutants. The dcl3-1and rdr2-1 mutants lack chromatin RNAi-associated, 24-nucleotide siRNAs,dcl2-1 and rdr1-1 mutants have defects in antiviral siRNA biogenesis,and the rdr6-15 mutant is defective in ta-siRNA biogenesis (Peragine etal., Genes & Dev 18:2369-2379, 2004; Vazquez et al., Mol Cell 16:69-79,2004b; Xie et al., PLoS Biol 2:642-652, 2004). The rdr6-15 mutantcontains a new insertion allele, but displays most of the sameproperties of previously characterized rdr6 mutants (Allen et al., NatGenet. 36:1282-1290, 2004). Specifically, rdr6-15 plants displays rapidjuvenile-to-adult phase change and accompanying morphological defects(FIG. 4A), and accumulates low levels of rdr6-15 transcript.

Expression profiling was done with triplicate biological samples onAffymetrix ATH1 arrays. Because DCL1, HEN1, HYL1, and likely HST, arerequired for miRNA biogenesis or function, we predicted that miRNAtarget genes would be upregulated coordinately in the correspondingmutants and largely unaffected in the siRNA biogenesis mutants. As agroup, previously validated and predicted target genes (Bin 1+2 genes)generally behaved as anticipated, although clearly not all genes wereupregulated in the miRNA mutants (FIG. 4B). Of the 81 genes present onthe ATH1 array, 27 were significantly (P<0.01, ANOVA) upregulated in twoor more of the miRNA mutants, although only 16 genes were significantlyupregulated in all four miRNA mutants. Transcripts for MYB101 (miR159target At2g32460) and a NAC domain gene (miR164 target At5g61430) weresignificantly (P<0.01, ANOVA) downregulated in the miRNA mutants,suggesting they may be negatively regulated by a factor that is undermiRNA control. Targets from Bins 3+4, of which only 12 were representedon the array, were generally upregulated in the miRNA mutants butunaffected by the siRNA mutants, although the At2g39680 transcript(antisense to validated miR173 target) was significantly upregulated inrdr6-15 as well as in miRNA-defective mutant plants (FIG. 4C). Inaddition, a list of genes that were affected (P<0.01, ANOVA) in each ofthe dcl1-7, hen1-1 and rdr6-15 mutants was generated. This listcontained five genes [At4g29770, At2g39680, At5g60450 (Auxin ResponseFactor4, ARF4), At2g33860 (ARF3) and At1g12770], all of which wereup-regulated in the three mutants (FIG. 4D). These genes were predictedto be either miRNA targets that were also subject to a RDR6-dependentRNAi pathway, or ta-siRNA targets. Three of these genes were shown toyield transcripts that function as ta-siRNA targets (At4g29770, ARF3 andARF4), one a predicted ta-siRNA target (At1g12770), and one a novel typeof miRNA target (At2g39680).

To analyze the variation patterns among all predicted and validatedmiRNA targets, two analyses were done. First, a Principal ComponentsAnalysis (PCA) was done using expression data from Bins 1-4. Aneigenvector that accounted for 65% of the variation among conditionsrevealed that the miRNA mutants were unified as havingtarget-upregulation effects, and the siRNA mutants were unified ashaving no effects (FIG. 4E). No other eigenvector accounted for morethan 9% of the variation. Among 30 genes highly correlated to theprimary eigenvector (r>0.95), 6 were validated targets, plus one 2PGKgene (At3g45090) closely related to the validated miR477 target. Thepredicted miR477 target site in At3g45090 failed the MFE ratio (0.69),although the expression profile suggests that At3g45090 is a miRNAtarget. Second, an unsupervised hierarchical clustering analysis wasdone, and correlated conditions were displayed as an expression tree.The four miRNA-defective mutants grouped within one clade, with dcl1-7and hen1-1 forming a subclade distinct from an hst-15/hyl1-2 subclade(FIG. 4F). The dcl1-1, dcl2-1, rdr1-1 and rdr2-1 mutants formed adistinct expression clade.

To compare more broadly the effects of miRNA and siRNA defects on theArabidopsis transcriptome, condition pairs were analyzed usingscatterplots. Also, a similar clustering analysis was done as fortargets, using all genes. Expression values (fold-change relative tocontrols) for genes that are coordinately affected in two mutants shouldremain on the diagonal, whereas genes that are differentially in twomutants affected fall above or below the diagonal. Based on thisapproach, the effects of hyl1-2 were most similar to the effects ofhst-15, and the effects of dcl1-7 were most similar to the effects ofhen1-1 (FIG. 4G). In contrast, there was little similarity betweentranscriptome-wide effects of any of the miRNA mutants and siRNAmutants, as exemplified by the hyl1-2/dcl3-1 comparisons (FIG. 4G).Among all conditions, the miRNA-defective mutants grouped within oneclade, and the siRNA mutants formed a distinct clade (FIG. 4F). With allgenes considered, the rdr6-15 mutant did not group with either miRNA- orsiRNA-defective mutants. Thus, with the major exceptions describedbelow, the expression profiling data indicate that miRNA-mediatedregulation of targets and downstream genes is largely independent of thesiRNA pathways.

miR173 Guides in-Phase Processing of Precursor Transcripts for Ta-siRNAsat Several Loci

Four miR173 targets were predicted based on the EST database but not theannotated transcript database. One of these predicted targets wasantisense relative to the annotated gene At2g39680. Two other miR173target sites were predicted based on ESTs AU235820 and CD534192 fromparalogous loci; a third paralogous locus also contained the conservedmiR173 site. miR173 target validation data for transcripts deriving fromeach of these four loci were obtained (FIG. 3B). None of the miR173target transcripts contained extended, conserved protein-codingsequences.

Inspection and analysis of the four loci yielding miR173-targetedtranscripts revealed that each was a confirmed or predictedta-siRNA-generating locus (FIG. 5). The three paralogous loci, termedTAS1a, TAS1b and TAS1c yielded siR255 and several similar sequences(siR289, siR752, and siR850, also referred to as siR289, siR752 andsiR850, respectively) in tandem, 21-nucleotide arrays. These ta-siRNAswere characterized previously and shown to require DCL1, RDR6, SGS3, andAGO1 (Peragine et al., Genes & Dev 18:2369-2379, 2004; Vazquez et al.,Mol Cell 16:69-79, 2004). siR255 (formally TAS1a 3′D6(+), TAS1b 3′D6(+),TAS1c 3′D3(+)) was shown to target transcripts from the related genesAt4g29760, At4g29770, and At5g18040 (functions unknown) for degradationin a manner similar to plant miRNAs. This was consistent with theexpression profiling data, in which At4g29770 was one of five genesup-regulated in dcl1-7, hen1-1, and rdr6-15 plants (FIG. 4D). The fourthmiR173 target locus, TAS2 (which was antisense to annotated At2g39680),possessed the hallmarks of a ta-siRNA-generating site, including thederivation of five cloned small RNAs representing both polarities inaccurate, 21-nucleotide register (FIG. 5C) and up-regulation in dcl1-7,hen1-1, and rdr6-15 plants (FIG. 4D). The TAS2 (At3g39680) locus mappedapproximately 2 kb away from, and in the same orientation as, TAS1cAt2g39675, raising the possibility that both ta-siRNA sets arise fromthe same precursor transcript (FIG. 5C). Relative to miRNAs, siR255 andsiR1511 small RNAs were relatively abundant as they corresponded to the19^(th) and 10^(th) most frequently cloned sequences, respectively, fromthe small RNA libraries in the ASRP database (Table 5).

TABLE 5 Highly represented small RNAs in the ASRP database Rank SmallRNA Family ASRP no. Total sequences 1 miR169 1430 25570 2 miR156 142314029 3 miR169 1751 6491 4 miR161.2 563 6227 5 miR160 1426 4752 6 miR1591425 4567 7 miR169 1514 3944 8 miR166 934 3482 9 miR167 5 2893 10siR1511 ta-siRNA 1511 1901 11 miR390 754 1373 12 miR169 1802 874 13miR169 1749 685 14 miR169 1761 660 15 miR168 1429 642 16 miR390 1703 58917 miR169 276 457 18 miR169 1757 405 19 siR255 ta-siRNA 255 321 20miR169 1775 299

To confirm that TAS2 is a ta-siRNA-generating locus, and to extend theanalysis of biogenesis requirements of this class of small RNA,TAS2-derived small RNAs and siR255 from the miRNA- and siRNA-defectivemutants were analyzed in blot assays. Small RNAs from the oppositestrand at the TAS2 locus were also analyzed. Accumulation of each smallRNA was lost or diminished in dcl1-7, hen1-1, hyl1-2, rdr6-11 andsgs3-11, but not in hst-15 (FIG. 5D). Accumulation levels wereunaffected in dcl2-1, dcl3-1, rdr1-1 and rdr2-1 mutants (FIG. 5D). Thesedata confirm that TAS2 is a ta-siRNA-generating locus.

The biogenesis data were consistent with a model in which ta-siRNAprecursor transcripts are recognized by RDR6/SGS3 and converted (atleast partially) to dsRNA forms, which are then processed by DCL1 inphased, 21-nucleotide intervals to form ta-siRNA duplexes. Setting thecorrect register must be a critical step in this pathway, asout-of-register processing would yield small RNAs with insufficientcomplementarity to their targets. We hypothesized that miR173-guidedcleavage of precursor transcripts generates a terminus that, afterRDR6/SGS3-dependent conversion to dsRNA, functions as a start point forsuccessive DCL1-mediated cleavage events in 21-nucleotide intervals.This hypothesis predicts that the predominant ta-siRNAs will form with a21-nucleotide phase starting at the miR173 cleavage site. A systematiccoding system, in which hypothetical DCL1 cleavage products from themiR173-targeted strand [3′D1(+), 3′D2(+), 3′D3(+), etc.] and oppositestrand [3′D1(−), 3′D2(−), 3′D3(−), etc.] were assigned a strict phasingrelative to miR173 target sites, was devised (FIG. 5A, B, C).

Each of the nine cloned ta-siRNAs identified collectively at the fourmiR173-targeted loci mapped precisely to the phasing interval set bymiR173-guided cleavage (FIG. 5A, B, C). As predicted from the knownproperties of Dicer-like enzymes, small RNAs from the non-targetedstrand (for example, siR143 and siR1946) were offset by two nucleotidesrelative to the complementary sequence on the target strand. Theregister was maintained at each locus through at least the 3′D6position, and at TAS1a through the 3′D8 position. A total of 19 uniquesmall RNAs, from positions 3′D1 to 3′D8, had 5′ ends formed by accuratein-phase cleavage but 3′ ends offset by one or two nucleotides. Slightvariation of this nature was expected, as Arabidopsis miRNA populationsfrequently contain processing variants that differ by one or a fewnucleotides. In addition to TAS1-derived siRNAs (e.g. siR255), whichwere confirmed to guide cleavage of mRNA targets (FIG. 5E), ahypothetical ta-siRNA from the 3′D6(−) position at the TAS2 locus waspredicted to interact with at least two PPR gene transcripts (At1g12770and At1g63130, FIG. 5E). At1g12770 was one of the five dcl1-1, hen1-1and rdr6-15-upregulated genes (FIG. 4D), which was consistent withidentity as a ta-siRNA target, although we were unable to validate acleavage site at the predicted position within the transcript (FIG. 5E).

miR390 Guides in-Phase Processing of Ta-siRNAs Regulating ARF3 and ARF4

The predicted target of miR390 was a transcript from the annotated geneAt3g17185 (FIG. 6A), for which no function was assigned previously. Thehypothetical protein encoded by this gene is small (50 residues) andcontains no recognizable motifs, raising the possibility that At3g17185is a misannotated, protein-noncoding locus. The miR390 target site wasvalidated by 5′RACE analysis (9/22 PCR products sequenced), although asecond cleavage site 33 nucleotides away was detected at approximatelythe same rate (11/22 PCR products).

The hypothesis that At3g17185 is a ta-siRNA-generating locus targeted bymiR390 was tested by analysis of small RNAs from the locus, andprediction and validation of putative ta-siRNA target genes. Twolow-abundance, cloned small RNAs from sequences to the 5′ side of themiR390 cleavage site were identified (FIG. 6A). siR1769 derivedprecisely from the 5′D1(+) position, whereas siR1778 was out-of-register(relative to the miR390-guided cleavage site) between the −5′D7 and 5′D8positions. Blot assays using strand- or sequence-specific radiolabeledprobes to detect small RNAs arising from between the 5′D5 to the 5′D11positions revealed that DCL1-, HEN1- and RDR6- and SGS3-dependent,21-nucleotide small RNAs arose from both strands (FIG. 6B). Thus, theAt3g17185 locus forms transcripts that yield small RNAs with biogenesisrequirements consistent with other ta-siRNAs. In addition to21-nucleotide RNAs, this locus also yielded detectable 24-nucleotideRNAs, which were clearly DCL3- and RDR2-dependent and RDR6- andSGS3-independent (FIG. 6B).

Potential targets of sequenced and hypothetical ta-siRNAs from theAt3g17185 locus were identified through several computational andexperimental validation steps. First, phylogenetic conservation of themiR390 target site, which was predicted to set the phasing for ta-siRNAprecursor processing, was analyzed. Transcripts and ESTs from each of 17species of monocot and dicot plants contained a miR390 target site,which was uniquely conserved relative to immediate flanking sequence ineach case (FIG. 6C). Second, functional ta-siRNAs and their targets werepredicted to be phylogenetically conserved across an equivalentevolutionary distance. In Arabidopsis, two highly conserved, tandem21-nucleotide sequences were detected at positions that nearlyco-aligned with the hypothetical 5′D7(+) and 5′D8(+) positions relativeto the miR390 cleavage site (FIG. 6C). These two intervals containednear-identical copies of the same sequence, which was conserved amongall transcripts that contained a miR390 target site (FIG. 6C). Thespacing between the conserved, tandem sequences and the miR390 targetsite varied between the 5′D7(+) and 5′D8(+) positions in differentspecies. In all plants, however, the tandem sequences and the miR390target site varied between the 5′D7(+)/5′D8(+) and the 5′D3(+))/5′D4(+)positions in different species. In all plants, however, the tandemsequences started in either perfect 21-nucleotide register (5/19species) or one-nucleotide offset (14/19 species) relative to the miR390cleavage site.

Third, using the rules developed for miRNA target prediction, four genes(ARF1, ARF2, ARF3, and ARF4) were predicted to be targets of theseconserved ta-siRNAs. Both ARF3 and ARF4 genes behaved as ta-siRNAtargets, as each was up-regulated in dcl1-7, hen1-1 and rdr6-15 mutantplants (FIG. 4D). Both ARF3 and ARF4 genes from 16 species contained tworegions (‘A’ and ‘B’) of complementarity to the predicted ta-siRNAs(FIG. 6D); the ‘A’ site was also conserved in ARF1 and ARF2 genes acrossall plant species tested. And fourth, the ‘A’ site in both ARF3 and ARF4was validated as a ta-siRNA target site by 5′RACE. In contrast to mostmiRNA target sites, the ARF3 and ARF4 ‘A’ site contained several minorcleavage products in addition to the product formed by cleavage at thecanonical target position (FIG. 6D). Evidence supporting ta-siRNAtargeting at the ‘B’ site within the ARF4 transcript was also obtained(FIG. 6D). Thus, the ta-siRNA-generating locus was named TAS3.

Although a small RNA from the TAS3 5′D2(−) position was not cloned, ahypothetical ta-siRNA from this position may account for the second TAS3transcript cleavage site mapped by 5′RACE (FIG. 6A). This cleavage siteoccurs precisely at the position predicted if TAS3 5′D2(−) guidedcleavage by a RISC-like mechanism. This cleavage site would also set thephase for ta-siRNA precursor processing to generate siR1778. Thissuggests that ta-siRNAs have the potential to interact with transcriptsfrom which they originate as well as mRNA targets.

Discussion

Combined with previous data, most notably from Jones-Rhoades et al., weare now aware of 25 validated miRNA families, 53 unique miRNA sequencesand 99 potential MIRNA loci in A. thaliana. Seventy-three genes have nowbeen validated experimentally as targets for miRNAs in 24 families.Fifty-three targets were validated in previous studies. Twenty predictedtargets of eleven miRNAs were validated or confirmed in this study (FIG.5, Table 3). These included mRNAs for SBP4 (miR156), Auxin ResponseFactor 16 (ARF16; miR160), two NAC domain proteins (miR164), AtHB15(miR165/166), ARF6 (miR167), six HAP2 family proteins (miR169), E2-UBC(miR399), AGO2 (miR403), 2PGK (miR447), and five non-coding genes(miR173 and miR390).

miRNAs are processed from genes that produce a primary transcript thatforms a stable foldback structure, processed by DCL1, and thereforerequires no polymerase and produces no antisense small RNAs.Trans-acting siRNAs have similar biogenesis requirements as miRNAs, butlack a stable foldback structure (Peragine et al., Genes & Dev18:2369-2379, 2004; Vazquez et al., Mol Cell 16:69-79, 2004b). As aresult, they require a polymerase, most likely RDR6, for second strandgeneration. Two defining characteristics of ta-siRNAs are the presenceof antisense 21-nucleotide small RNAs, and a linear, in-phase processingof both sense and small RNAs. Unlike other classes of siRNAs, ta-siRNAscan be incorporated into RISC and trigger site-specific cleavage oftarget genes, similar to miRNAs. Both miRNAs and ta-siRNAs are uniquelyinsensitive to DCL2, DCL3, RDR1, and RDR2. In the absence of acomprehensive profile of biogenesis mutants, it is impossible toproperly catalog small RNA function. Using this strict set of criteria,we characterized four miRNA families, two of which were previouslyidentified.

Our target prediction algorithm confirmed the robust predictions for themajority of validated miRNAs. Additional targets were validated withinthis group, including eight targets residing in the untranslated regionof the target messenger RNA, including SPL4, an E2-UBC gene At2g33770,and six HAP2 transcripts. Notably, most miR156 targets are located inthe coding region of SPL transcripts, whereas two reside immediatelydownstream of the stop codon in the 3′ UTR, SPL3 and SPL4 (Rhoades etal., Cell 110:513-520, 2002). Interestingly, two splicing variants ofSPL4 exist, one with the miR156 target site (AU227430, BP595743) and onethat lacks the target site (BX814070.1), although the coding sequence isunchanged. Potentially the alternately spliced variant of SPL4 wouldallow an additional level of miRNA-mediated control. The E2-UBC gene isunique in that it contains five miR399 targets in its 5′ UTR. Themultiple miR399 target sites are conserved among distantly related plantspecies. The multiple sites might be necessary for miRNA targeting inthe 5′ UTR to increase the chance of cleavage before ribosomes couldclear the miRNA from the mRNA, although the nature of multi-siteregulation remains to be determined.

We identified six novel miRNA target loci in the Arabidopsis ESTdatabase using a computational prediction algorithm developed based onvalidated miRNA-target characteristics. Previous computational searchesfor miRNA targets in plants have only used transcript databases, as aresult missing these target genes (Jones-Rhoades & Bartel, Mol Cell14:787-799, 2004). The miR403 target, Ago2, is the second Argonautefamily gene to be miRNA regulated. Arabidopsis Ago2 does not have aclose ortholog in mammals, and its role in small RNA function is unknown(Carmell et al., Genes Dev 16:2733-2742, 2002; Mochizuki et al., Cell110:689-699, 2002). The remaining five miRNA targets from the ESTdatabase search are non-protein coding loci, all of which produce21-nucleotide small RNAs, in phase with the miRNA cleavage site. Fourloci were validated to generate functional ta-siRNAs, including a familyof unclassified genes, as well as ARF3 and ARF4. The ta-siRNA targetgenes were upregulated in dcl1-7, hen1-1, and rdr6-15, which couldprovide a diagnostic test for ta-siRNA target genes. Both miR390 and theTAS3 locus are conserved among distantly related plants. A completeprofile of small RNA coding-genes will require thorough complementarymolecular and computational approaches, perhaps with consideration ofconserved 21-nucleotide regions in annotated intergenic regions.Potentially, identification of non-protein coding genes will befacilitated by genome tiling data (Yamada et al., Science 302:842-846,2003) in combination with small RNA cloning and biogenesis profiling.

We propose a model in which miRNA cleavage initiates the starting phasefor ta-siRNA production (FIG. 7). The primary miRNA targeted cleavage ofan RNA Polymerase II transcript (step 1) recruits a RISC complex to theRNA. In addition, RDR6 and SGS3 could be recruited by theRISC:miRNA:target complex. Cleavage by the miRNA at a specific positioncreates a unique initiation position. Following cleavage, RDR6/SG83polymerize a second strand (step 2), creating a double stranded RNA(dsRNA Either the 5′ (e.g. TAS3) or 3′ (e.g. TAS1 and TAS2) cleavageproduct can be utilized as the RDR6 template. In either case, DCLprocessing of 21-nucleotide siRNA duplexes (step 3) proceeds in-phasefrom the primary miRNA cleavage site. Dicer in animals is known tocatalyze cleavage from a free end (Zhang et al., Cell 118:57-68, 2004).We did not identify any in-phase small RNAs beyond nine phases from themiRNA cleavage initiation site, suggesting either the RDR6/SG83 complexor the DCL1 complex is not highly processive. One strand of the siRNAduplex is loaded back into a RISC complex, following the known siRNAincorporation rules (Khvorova et al., Cell 115:209-216, 2003; Schwarz etal., Cell 115:199-208, 2003). Following RISC incorporation of theta-siRNA (step 4), ta-siRNAs function like miRNAs to facilitate cleavageor target genes in trans (step 5).

The regulatory role of miRNAs for all target genes previously identifyis to repress target gene expression, through either cleavage or byblocking translation. Our results suggest that miRNAs also act as apositive regulator of ta-siRNA biogenesis through recruitment of RISCand initiation of unique and highly specific phasing for DCL1-mediatedprocessing. Although we have only found evidence for a single activeta-siRNA (or highly similar tandem sequence repeat), multiple, phasedta-siRNAs could provide an advantage through generation of multiple,independent regulatory (ta-siRNA-forming) units from a single locus. Thediscovery that a miRNA:ta-siRNA:target regulon is conserved amongdistantly related plants shows that this type of regulation is notspecific to Arabidopsis, opening the possibility of an entirely newclass of small RNA mediated gene regulation.

Example 2 MiRNA-Directed Biogenesis of Ta-siRNAs In Vivo

To experimentally test the hypothesis that ta-siRNA biogenesis isinitiated by miRNA-guided cleavage of primary transcripts, TAS1 and TAS2were co-expressed transiently with MIR173 in Nicotiana benthamiana. IfmiR173 is required for siR255 production, as predicted herein, thensiR255 should be formed only in the presence of miR173. At least some ofthe material in this example was published in Allen et al. (Cell121:207-221, 2005), which is incorporated herein by reference in itsentirety.

Expression cassettes containing the TAS1a, TAS1b, TAS1c and TAS2 loci(which all include both an initiator sequence, containing an initiatorcleavage site, and a gene suppressing element) were delivered intoNicotiana benthamiana plant cells (Llave et al., Plant Cell14:1605-1619, 2002; Palatnik et al., Nature 425:257-263, 2003) in thepresence or absence of an expression cassette containing miR173, andta-siRNA accumulation was scored. Expression of full-length TAS1b[35S:TAS1b(+)], a short version of TAS1b [35S:TAS1b(+)sh], andfull-length TAS1a [35S:TAS1a(+)] resulted in siR255 accumulation only inthe presence of a construct (35S:miR173) expressing miR173 (FIG. 14A,lanes 7, 8, 13, 14, 17, 18). Likewise, siR255 from the TAS1c construct[35S:TAS1c(+)], and siR1511 from the TAS2 construct [35S:TAS2(+)], bothaccumulated only in the presence of the miR173 construct (FIG. 14B,lanes 7, 8, 11, 12). Ta-siRNAs were not detected after expression of anyof the TAS1 or TAS2 constructs alone (FIG. 14A, lanes 3, 4, 11, 12, 15,16; FIG. 14B, lanes 5, 6, 9, 10), or after expression of themiR173-non-targeted strand of the short version of TAS1b[35S:TAS1b(−)sh] in either the presence or absence of miR173 (FIG. 14A,lanes 5, 6, 9, 10). In the presence of miR173, siR255 accumulated tolevels up to 7.6 fold higher using the TAS1a(+) and TAS1c(+) constructscompared to the TAS1b(+) constructs. This may reflect a relatively poormiR173-TAS1b interaction, which involves two mismatched positions nearthe target cleavage site (FIG. 5B).

To confirm that ta-siRNA biogenesis requires miRNA-directed targeting ofprimary transcripts, a TAS1b mutant construct [35S:TAS1b(+)shmut1] witha disrupted miR173 target site was expressed in the presence of miR173.The TAS1b mutant was also expressed in the presence of a modified miR173construct (35S:miR173res1) containing base substitutions to restoreinteraction with the TAS1b mutant (FIG. 14C, top). Mutations affectingthe TAS1b target site or miR173 resulted in the loss of siR255biogenesis (FIG. 14C, lanes 7, 8, 11, 12). In contrast, siR255accumulation was restored when the TAS1b mutant was co-expressed withthe miR173res1 construct (FIG. 14C, lanes 13, 14).

Thus, in each independent experiment, siRNAs from each locus weredetected (by RNA blot assay) only in the presence of a construct thatformed miR173 (FIG. 14). Mutations that disrupted the miR173 target sitein the TAS1b construct eliminated siRNA (siR255) formation. However,mutations in the miR173 sequence to restore complementarity with themutated target sequence restored the formation of siR255 (FIG. 14).These data support the model that states ta-siRNA biogenesis requires amiRNA-guided initiation cleavage. It also demonstrates that anexpression cassette containing an initiator sequence and a genesuppressing element can direct production of a siRNA in the presence ofan expression cassette containing a miRNA. Stated another way, thesedata show that a functional miRNA target site in the ta-siRNA primarytranscript is required to trigger ta-siRNA formation.

See also Example 6, below, for additional details.

Example 3 Plant Transformation Vectors/Plasmids

This example illustrates the construction of plasmids for transferringrecombinant DNA into plant cells which can be regenerated intotransgenic plants, e.g., expressing in a plant siRNA for suppression ofan endogenous gene. See also Example 6, below.

A recombinant DNA construct for plant transformation construct 1A isfabricated for use in preparing recombinant DNA for transformation intocorn tissue comprising the a selectable marker expression cassette, asiRNA-triggering cassette and a cleavage initiating cassette. The markerexpression cassette comprises a rice actin 1 promoter element(s)operably linked to sequence(s) encoding a chloroplast transit peptidefrom Arabidopsis thaliana ShkG gene and an aroA protein fromAgrobacterium tumefaciens, strain CP4, followed by a 3′ region of anAgrobacterium tumefaciens nopaline synthase gene (nos). ThesiRNA-triggering cassette is positioned tail to tail with the markerexpression cassette and comprises 5′ regulatory DNA from a maize seedspecific promoter L3 (as disclosed in U.S. Pat. No. 6,433,252) operablylinked to DNA encoding RNA comprising an initiator sequence that ishighly complementary to a microRNA such as miR173 (or any microRNA orsiRNA, including any listed herein) and at least one 21-nucleotidesegment from LKR. An initiation cleavage cassette is positioned head tohead with the marker expression cassette and comprises a maize seedspecific promoter L3 and DNA expressing a microRNA (e.g., miR173) thatguides cleavage of the initiation cleavage site in the siRNA-triggeringcassette. Construct 1A is useful for plant transformation, e.g. bymicroprojectile bombardment. Transgenic corn callus is produced bymicroprojectile bombardment of construct 1A using methods disclosed inU.S. Pat. No. 6,399,861.

A plasmid vector 1B for use in Agrobacterium-mediated methods of planttransformation is prepared by inserting construct 1A into a plasmidbetween left and right T-DNA border sequences from Agrobacterium.Outside of the T-DNA borders the plasmid also contains origin ofreplication DNA to facilitate replication of the plasmid in both E. coliand Agrobacterium tumefaciens and a spectinomycin/streptomycinresistance gene for selection in both E. coli and Agrobacterium.Transgenic corn callus is produced by Agrobacterium-mediatedtransformation of plasmid vector 1B using methods disclosed in U.S. Pat.No. 5,591,616.

Transgenic corn plants are regenerated from transgenic callus producedby microprojectile bombardment and Agrobacterium-mediatedtransformation; callus is placed on media to initiate shoot developmentin plantlets which are transferred to potting soil for initial growth ina growth chamber at 26° C. followed by growth on a mist bench beforetransplanting to 5 inch pots where plants are grown to maturity. Theplants are self fertilized and seed is harvested for screening as seed,seedlings or progeny R2 plants or hybrids, e.g. for yield trials in thescreens indicated above. Transgenic plants with higher levels of lysineresulting from suppressed levels of LKR and which are homozygous for therecombinant DNA are identified. The homozygous plants are selfpollinated to produce transgenic seed with the recombinant DNAcomprising siRNA-triggering cassettes.

Example 4 Inhibition of Plant Pest Genes

This example illustrates the construction of plasmids for transferringrecombinant DNA into plant cells which can be regenerated intotransgenic described herein, particularly expressing in a plant siRNAfor suppression of genes in a plant pest.

Recombinant DNA constructs 2A, 2B and 2C are fabricated for soybeantransformation by microprojectile bombardment essentially like construct1A except that the promoter used in the siRNA-triggering cassette andthe initiation cleavage cassette is a root tissue-expressing promoterand the 21-nucleotide segment is derived from DNA encoding soybean cystnematode proteins as disclosed in US Patent Application Publication2004/0098761 A1. In construct 2A the 21-nucleotide segment is from amajor sperm protein; in construct 2B the 21-nucleotide segment is from achitin synthase; and in construct 2C the 21-nucleotide segment is froman RNA polymerase II. Soybean is transformed by microprojectilebombardment using constructs 2A, 2B and 2C using methods as disclosed inU.S. Pat. No. 5,914,451 and transgenic soybean plants are regeneratedwhich exhibit resistance to soybean cyst nematode infestation ascompared to control plants.

Plasmid vectors 2D, 2E and 2F for use in Agrobacterium-mediated methodsof plant transformation are prepared by inserting constructs 2A, 2B and2C, respectively, into plasmids with T-DNA borders similar to plasmidvector. Soybean is transformed by Agrobacterium-mediated transformationof plasmid vectors 2D, 2E and 2F using methods disclosed in U.S. Pat.No. 6,384,301 and transgenic soybean plants are regenerated whichexhibit resistance to soybean cyst nematode infestation as compared tocontrol plants.

Example 5 Expression of Arabidopsis thaliana MIRNA Genes

Recent molecular cloning and computational analyses have identifiednearly one-hundred potential genetic loci for MIRNA genes in theArabidopsis thaliana genome. However, information about the structureand expression of these genes is generally lacking. The transcriptionalstart site for each of 63 miRNA precursor transcripts from 52 MIRNA (99total loci tested) was mapped. A portion of the loci yielded multipletranscripts from alternative start sites, and some contained intronsbetween the foldback structure and the 5′ end. Analysis of arepresentative set of transcripts revealed characteristics consistentwith transcription by Pol II. A canonical TATA box motif was identifiedcomputationally upstream of the start site(s) at some MIRNA loci. The 5′mapping data were combined with miRNA cloning and 3′-PCR data todefinitively validate expression some of known MIRNA genes. These dataprovide a molecular basis to explore regulatory mechanisms of miRNAexpression in plants.

Material from this example was published as Xie et al., Plant Physiol.138(4):2145-2154, 2005; Epub 2005 Jul. 22, which is incorporated hereinby reference in its entirety.

MicroRNAs (miRNAs) are ˜21-nucleotide noncoding RNAs thatpost-transcriptionally regulate expression of target genes inmulticellular plants and animals (Bartel, Cell 116:281-297, 2004).Mature miRNAs are generated through multiple processing steps fromlonger precursor transcripts that contain imperfect foldback structures.In animals, MIRNA genes are transcribed by RNA polymerase II (pol II)(Bracht et al., RNA 10:1586-1594, 2004; Cai et al., RNA 10:1957-1966,2004; Lee et al., EMBO J. 23:4051-4060, 2004), yielding a primarytranscript (pri-miRNA) that is processed initially by nuclearRNaseIII-like Drosha (Lee et al., Nature 425:415-419, 2003). Theresulting pre-miRNA transcripts are transported to the cytoplasm andprocessed by Dicer to yield mature-size miRNAs (Lee et al., EMBO J.21:4663-4670, 2002). Less is known about the miRNA biogenesis pathway inplants, although most or all miRNAs require Dicer-like1 (DCL1) (Park etal., Curr Biol 12:1484-1495, 2002; Reinhart et al., Genes Dev16:1616-1626, 2002). The lack of a Drosha ortholog in plants, and thefinding that DCL1 functions at multiple steps during biogenesis ofmiR163, suggest that the plant miRNA pathway may differ from the animalpathway (Kurihara & Watanabe, Proc Natl Acad Sci USA 101:12753-12758,2004). MiRNAs in both animals and plants incorporate into an effectorcomplex known as RNA-induced Silencing Complex (RISC) and guide eithertranslation-associated repression or cleavage of target mRNAs (Bartel,Cell 116:281-297, 2004).

Computational and molecular cloning strategies revealed over 100potential MIRNA genes belonging to at least 27 families in theArabidopsis genome (Llave et al., Plant Cell 14:1605-1619, 2002; Metteet al., Plant Physiol 130:6-9, 2002; Park et al., Curr Biol12:1484-1495, 2002; Reinhart et al., Genes Dev 16:1616-1626, 2002;Jones-Rhoades & Bartel, Mol Cell 14:787-799, 2004; Sunkar & Zhu, PlantCell 16:2001-2019, 2004; Wang et al., Genome Biol 5:R65, 2004). ThesemiRNA families target mRNAs encoding proteins that include a variety oftranscription factors involved in development, DCL1 and the RISC factorARGONAUTE1(AGO1), components of the SCF complex involved inubiquitin-mediated protein degradation, and several other classes ofmetabolic and stress-related factors (Rhoades et al., Cell 110:513-520,2002; Xie et al., Curr Biol 13:784-789, 2003; Jones-Rhoades & Bartel,Mol Cell 14:787-799, 2004; Sunkar & Zhu, Plant Cell 16:2001-2019, 2004;Vaucheret et al., Genes Dev 18:1187-1197, 2004) (see also Example 1).Based on tissue distribution and limited in situ expression data, mostplant miRNAs are likely regulated at spatial and/or temporal levelsduring development (Chen, Science 303:2022-2025, 2004; Juarez et al.,Nature 428:84-88, 2004; Kidner & Martienssen, Nature 428:81-84, 2004).Overexpression or knockout of MIRNA genes, or expression of MIRNA genesoutside of their normal expression domains, can lead to severedevelopmental defects (Aukerman & Sakai, Plant Cell 15:2730-2741, 2003;Palatnik et al., Nature 425:257-263, 2003; Achard et al., Development131:3357-3365, 2004; Chen, Science 303:2022-2025, 2004; Juarez et al.,Nature 428:84-88, 2004; Kidner & Martienssen, Nature 428:81-84, 2004;Laufs et al., Development 131:4311-4322, 2004; Mallory et al., Curr Biol14:1035-1046, 2004a; Mallory et al., EMBO J. 23:3356-3364, 2004; McHale& Koning, Plant Cell 16:1730-1740, 2004; Emery et al., Curr Biol13:1768-1774, 2003; Zhong & Ye, Plant Cell Physiol 45:369-385, 2004).Understanding the mechanisms governing MIRNA gene expression patternsand integration into regulatory networks will be necessary for a clearunderstanding of the biological function of miRNAs.

In this example, several new Arabidopsis miRNAs were identified by acomputationally assisted cloning approach and the use of mutants thatcontained miRNA-enriched pools of small RNAs. Expression of 99 MIRNAgenes in Arabidopsis was examined experimentally. First, featuresassociated with transcription initiation of MIRNA genes were analyzed,revealing core promoter, start sites and other properties that wereconsistent with a pol II mechanism of transcription. And second, asurvey of expression of each known MIRNA locus was done to identifyfunctional MIRNA genes.

Materials and Methods

Cloning of A. Thaliana Small RNAs and miRNA Prediction.

Extraction of low molecular weight RNA and library construction was doneas described (Llave et al., Plant Cell 14:1605-1619, 2000; Lau, Science294:858-862, 2001). RNA was extracted from three-day post germinationseedlings, embryos from developing siliques, aerial tissues includingrosette leaves and apical meristems, or stage 1 to 12 enrichedinflorescence from wildtype Columbia-0, and jaw-D, rdr2-1 and dcl3-1mutants described previously (Palatnik et al., Nature 425:257-263, 2003;Xie et al., PLoS Biol 2:642-652, 2004). Seedling libraries wereconstructed for Col-0, rdr2-1, and dcl3-1, embryo libraries for rdr2-1,aerial libraries for jaw-D, and inflorescence libraries for Col-0 andrdr2-1. Sequences were filtered to remove organellar, rRNA, and thosenot present in A. thaliana. Remaining small RNAs between 18 and 26nucleotides were deposited in the ASRP database (available on-line atasrp.cgrb.oregonstate.edu/). Candidate miRNA prediction used a set ofsix filters. First, structural RNAs were filtered before entry into theASRP database by manual scoring of BLAST hits to known rRNA, tRNA, andorganellar RNA. Second, small RNAs from repeats identified usingRepeatMasker (Jurka, Trends Genet. 16:418-420, 2000) or from predictedprotein-coding genes and pseudogenes only were removed. Third, a smallRNA cluster filter was applied to remove small RNAs within 500 nt ofanother small RNA in the opposite orientation. The fourth filter removedany small RNA outside the typical size (20-22 nucleotides). Fifth,characteristics including the minimum paired bases of the miRNA:miRNA*duplex in the reference set (≧16), maximum foldback size (350nucleotides), and a requirement for the miRNA and its duplex to be on asingle stem were determined. Foldbacks in which the miRNA:miRNA duplexcontained more than three contiguous unpaired bases were excluded. TheRNAFold in the Vienna RNA Package was used to predict potential duplexescontaining the small RNA, and those with duplexes not meeting the abovecriteria were excluded (Hofacker, Nucleic Acids Res 31:3429-3431, 2003).Sixth, validated miRNAs and closely related family members, as well assmall RNAs processed from a miRNA locus (including miRNA*) wereidentified by FASTA and comparison of small RNA loci on the ASRP genomebrowser. These small RNAs were annotated as family members of validatedmiRNAs, and removed from the predicted miRNA pool.

Small RNA Blot Analysis.

Low molecular weight RNA (5 μg) from A. thaliana inflorescence tissuewas used for miRNA and endogenous siRNA analysis. Mutant lines fordcl1-7, dcl2-1, dcl3-1, rdr1-1, rdr2-1, hen1-1, hyl1-2, rdr6-11,rdr6-15, and sgs3-11 were described previously (Park et al., Curr Biol12:1484-1495, 2002; Allen et al., Nat Genet. 36:1282-1290, 2004;Peragine et al., Genes & Dev 18, 2368-2379, 2004; Vazquez et al., CurrBiol 14:346-351, 2004; Xie et al., PLoS Biol 2:642-652, 2004). Thehst-15 allele used was the SALK_(—)079290 T-DNA insertion line fromABRC, which contains a T-DNA at position 1584 from the start codon.Probes for miR159, miR167, and AtSN1-siRNA blots were describedpreviously (Llave et al., Plant Cell 14:1605-1619, 2002; Zilberman etal., Science 299:716-719, 2003). All other miRNAs were detected usingend-labeled DNA oligonucleotides. Probes for ta-siRNA loci were PCRamplified from Col-0 genomic DNA, cloned into pGEMT-Easy, and verifiedby sequencing. Radiolabeled probes incorporating ³²P-UTP were made by T7RNA polymerase transcription, to obtain strand specific small RNAprobes. Probes were as follows: At1g17185 locus, Chr3:5862146-5862295;At2g39680 locus, Chr2:16546831-16547300.

5′RACE Mapping of MIRNA Transcripts

Two Arabidopsis thaliana (Col-0) sample preparations were used for RNAisolation: inflorescence tissues from 4-week old plants grown undergreenhouse condition and 4-day old seedlings grown on MS media in agrowth chamber. Total RNA was extracted using TRIzol reagent(Invitrogen) followed by column purification using a RNA/DNA midi kit(Qiagen). The extracts were subjected to two rounds of purificationusing Oligotex (Qiagen) for the enrichment of poly(A)⁺ RNA. The 5′ endsof MIRNA transcripts were mapped by a RNA ligase-mediated 5′RACE(RLM-5′RACE, Invitrogen). Complementary DNA (cDNA) was synthesized withpoly(A)+-enriched RNA (125 ng/reaction), which was first treated withcalf intestine phosphatase and tobacco acid pyrophosphatase (CIP+TAP),using random oligonucleotide hexamers as primers. A cDNA pool containingequal amounts of cDNA from each tissue was used as template in 5′RACEPCR with a primer (Invitrogen) specific to the RNA adaptor sequence anda locus-specific reverse primer. In cases where no product was detected,a second-round PCR was done using a 5′ nested primer and alocus-specific nested primer. The default annealing temperature in thetouchdown PCR reaction was 65° C. For a MIRNA locus with a negative5′RACE result after the second-round PCR, two additional PCR reactionswith the nested primers were done with altered annealing temperatures.The PCR products from a positive 5′RACE were gel-purified and clonedinto pGEM-Teasy vector. A minimum of 6 clones were sequenced for eachPCR product.

The RLM-5′RACE procedure was used to analyze the presence or absence ofa cap structure on several miRNA transcripts. A capped mRNA[Scarecrow-like6-IV (SCL6-IV)] and a non-capped RNA (miR171-guidedcleavage product of SCL6-IV mRNA) were used as control RNAs. ParallelRLM-5′RACE reactions were done using poly(A)+-enriched RNA that wasCIP+TAP treated and non-treated, which was selective for amplificationof 5′ ends that contained or lacked a cap structure, respectively.

For some miRNA transcripts, 3′RACE was done using poly(A)+-enriched RNA.cDNA was synthesized using an adaptor-tagged oligo(dT) primer. Twogene-specific forward primers were designed for each locus tested. Theidentity of the 3′RACE products were confirmed by sequencing. Thesequences of the locus-specific primers are provided in SEQ ID NOs: 349to 614, and were published in Supplementary Table 2 in Xie et al., PlantPhysiol. 138(4):2145-2154, 2005; Epub 2005 Jul. 22.

Computational Identification of Conserved Upstream Sequence Motifs

A 60-bp (−50 to +10) genomic sequence flanking the start site for 63transcripts from 47 MIRNA loci was analyzed using BioProspector, a Gibbssampling-based motif-finding program (Liu et al., 2004). Searches with amotif width of 6-8 nucleotides were done. In all cases, TATA-likesequences were identified as the only conserved motif. A second search(8-nucleotide width) was done using an extended MIRNA upstream region(−200 to +50) to analyze the distribution of the putative TATA motifusing MotifMatcher, with the 8-nucleotide motif matrix generated byBioProspector as a sample motif (Ao et al., Science 305:1743-1746,2004). Up to three matches to the TATA motif were allowed.

Results and Discussion

Identification and Validation of Arabidopsis miRNAs

Several small RNA libraries were constructed from wild-type (Col-0) A.thaliana seedling and inflorescence tissues, and from aerial tissues ofjaw-D plants that over express miR-JAW (miR319) (Palatnik et al., Nature425:257-263, 2003). Among all 2357 sequences analyzed collectively fromthese libraries, only 32.7% corresponded to known or subsequentlyvalidated miRNA families. Most of the remaining small RNAs correspondedto diverse sets of endogenous small RNAs arising from repeated sequencessuch as transposons, retroelements, simple sequence repeats, invertedduplications, rDNA genes and other genic and intergenic sequences (Llaveet al., Plant Cell 14:1605-1619, 2002; Xie et al., PLoS Biol 2:642-652,2004). To genetically enrich for miRNAs, small RNA libraries wereconstructed from embryo, seedling, and inflorescence tissues of rdr2-1mutant plants, and from seedlings of dcl3-1 mutant plants. These plantscontain relatively low levels of ˜24-nucleotide siRNAs from repeatedsequences, but maintain normal levels of miRNAs (Xie et al., PLoS Biol2:642-652, 2004). Among 3164 sequences analyzed collectively from therdr2-1 and dcl3-1 libraries, 70.5% corresponded to previouslycharacterized miRNAs, representing a 2.2-fold overall enrichmentrelative to the wild-type libraries. Endogenous siRNAs from known repeatfamilies (identified from RepBase) were reduced 43.9-fold in the mutantlibraries. The majority of the remaining small RNAs corresponded tosequences from two rdr2-independent small RNA-generating loci, or fromrRNA genes. Unique miRNA and endogenous siRNA sequences from alllibraries are available in the Arabidopsis Small RNA Project (ASRP)database (available on-line at asrp.cgrb.oregonstate.edu).

To identify new miRNAs in the cloned libraries, the small RNA sequenceswere subjected to a series of five computational filters (FIG. 8A). Thefilters were designed using the properties of a founder set ofpublished, validated Arabidopsis miRNAs with codes within the range ofmiR156-miR399 (excluding miR390 and miR391; RFAM). Among the 48 uniquemiRNA sequences from 92 loci (22 validated miRNA families) in thefounder set, 34 miRNA sequences from 71 loci (19 families) were in thecloned database. The initial filters eliminated all small RNA sequencesderiving from structural RNA genes, other annotated genes and repetitiveloci identified by RepeatMasker (FIG. 8A). Sequences originating fromloci that yielded multidirectional clusters of small RNAs, which is ahallmark of many siRNA-generating loci, were eliminated. Small RNAs thatwere not 20-22 nucleotides in length, based on the cloned sequence, werealso removed Small RNAs originating from loci that lacked the potentialto form a miRNA precursor-like foldback structure, consisting of a stemin which 16 or more positions within the putative miRNA-miRNA* duplexregion were paired, were excluded. To test the sensitivity of thesefilters, the complete founder set of miRNAs was processed through thefive filters. All but three passed, corresponding to a false negativerate of 0.032. MiR163 failed because it is 24 nucleotides long, andmiR166 from two loci failed because of 6 mispaired miRNA positionswithin the foldback stem. From the cloned dataset, a total of 103 smallRNAs from passed the five filters (FIG. 8A). These did not correspond to103 unique loci, however, as many miRNA-generating loci yield multipleprocessed forms that are offset by one or a few nucleotides Eliminationof all sequences corresponding to founder miRNAs yielded a set of 18small RNAs, corresponding to 13 genetic loci, as candidate new miRNAs(FIG. 8A). This set included miR390, miR391, miR403 and miR447 (FIG.8B). Six of the 18 small RNAs corresponded to a cluster of processingvariants from the two miR390 loci.

Given the high sensitivity of the computational filters using thefounder set, a second set of published Arabidopsis sequences with miRNAdesignations were analyzed. These have not been subjected to extensiveexperimental validation as miRNAs. This set includes all sequences withcodes between miR400-miR420 (Sunkar & Zhu, Plant Cell 16:2001-2019,2004; Wang et al., Genome Biol 5:R65, 2004), except miR403. In contrastto the founder set, most of the small RNAs in the second set failed atone or more steps. Six small RNAs (miR401, 405a-d, 407, 416) wereidentified as transposon-derived, two (miR402, 408) were from annotatedgenes, and ten (miR401, 404, 406, 408, 413, 414, 417-420) failed thefoldback prediction criteria. Given the high computational failure rate(0.84) of this set, which was 26-fold higher than the false negativerate of the founder set, it is likely that many or most of these areendogenous siRNAs and not bona fide miRNAs.

Candidate miRNAs from each of the 13 loci identified in thecomputational analysis was subjected to validation blot assays using aseries of Arabidopsis miRNA-defective (dcl1, hyl1, hen1, and hst) andsiRNA-defective (dcl2, dcl3, rdr1, rdr2, rdr6 or sgs3) mutants (Reinhartet al., Genes Dev 16:1616-1626, 2002; Kasschau et al., Dev Cell4:205-217, 2003; Jones-Rhoades & Bartel, Mol Cell 14:787-799, 2004;Vazquez et al., Curr Biol 14:346-351, 2004; Xie et al., PLoS Biol2:642-652, 2004). In addition, small RNAs were analyzed in transgenicplants expressing three viral RNAi suppressors (P1/HC-Pro, p19 and p21),which frequently enhance the level of miRNA accumulation (Mallory etal., Proc Natl Acad Sci USA 99:15228-15233, 2002; Kasschau et al., DevCell 4:205-217, 2003; Papp et al., Plant Physiol 132:1382-1390, 2003;Chapman et al., Genes Dev. 18:1179-86, 2004) but decrease the level ofta-siRNA accumulation. Previously validated miR159, miR167 and miR173,and AtSN1-derived siRNAs were analyzed in parallel as controls.Reproducible signals were detected in Col-0 and La-er control plantsonly using probes for miR390, miR391, miR403 and miR447 (FIG. 8C). Eachof these accumulated to relatively low levels in the dcl1-7, hen1-1 andhyl1-2 mutants, but accumulated to normal or near-normal levels in thedcl2-1, dcl3-1, rdr1-1, rdr2-1, rdr6-11 and sgs3-11 mutants (FIG. 8C,D).The hst-15 mutant accumulated nearly normal amounts of the fourcandidates as well as the three miRNA controls (FIG. 8C), indicatingthat miRNA accumulation in the tissues tested was relatively insensitiveto loss of HST function. MiR390, miR391, miR403 and miR447 were eitherup-regulated or unaffected by each of the three viral suppressorproteins (FIG. 8D). Based on structural and biogenesis criteria, weconclude that miR390, miR391, miR403 and miR447 are bona fide miRNAs.Small RNAs from the remaining eight loci (Table 6) were not detected inblot assays and were not characterized further.

TABLE 6 Predicted miRNA candidates tested  experimentally miRNA miRNAASRP valida- name, Locus no. Sequence tion notes  1,   754^(a)AAGCUCAGGAGGGAUAGCGCC yes miR390  2 SEQ ID NO: 143  3 1728UUCGCAGGAGAGAUAGCGCCA yes miR391 SEQ ID NO: 144  4  359AUUAGAUUCACGCACAAACUCG yes miR403 SEQ ID NO: 145  5 1890UUGGGGACGAGAUGUUUUGUUG yes miR447 SEQ ID NO: 146  6  382GAGCCGACAUGUUGUGCAACUU no not  SEQ ID NO: 147 detected  7  991AAUGGAAGCCUUGUCAGCUUAU no not  SEQ ID NO: 148 detected  8 1072UAAAGUCAAUAAUACCUUGAAG no not  SEQ ID NO: 149 detected  9 1345UAUAAGCCAUCUUACUAGUU no not  SEQ ID NO: 150 detected 10 1744UUCUGCUAUGUUGCUGCUCAUU no not  SEQ ID NO: 151 detected 11 1928UCUAAGUCUUCUAUUGAUGUUC no not  SEQ ID NO: 152 detected 12 1943CUGUCUUCUCAACUUCAUGUGA no not  SEQ ID NO: 153 detected 13 2028CGGCUCUGAUACCAAUUGAUG no not  SEQ ID NO: 154 detected ^(a)Fourprocessing variants from the two miR390 loci were cloned

MiR390 and miR391 are related miRNAs that differ by five nucleotides,whereas miR403 and miR447 are distinct from all other known miRNAs. IfmiR390 and miR391 are assigned to the same family, then Arabidopsiscontains 25 experimentally validated families of miRNAs encoded by up to99 genes (Table 7). Among these families, 19 are conserved betweendicots and monocots. One family (miR403) is conserved among familieswithin dicots, and five families (miR158, miR161, miR163, miR173 andmiR447) have been identified only in Arabidopsis.

TABLE 7 Arabidopsis miRNA families miRNA ASRP SEQ fami- miRNAlibrary^(b) Plant Target ID lies family Locus Sequence^(a) Col-0rdr2/dcl3 species^(c) family NO. 1 miR156 a-f UGACAGAAGAGAGUGAGCAC + +At, Bn, Gm,   SBP 155 Ha, Hv, Lj,   Mt, Nt, Os, Pta, Ptr,  Sb, Si, So,  St, Vv, Zm miR156 g CGACAGAAGAGAGUGAGCACA − − At 156 miR156 hUUGACAGAAGAAAGAGAGCAC − − At 157 miR157 a-d UUGACAGAAGAUAGAGAGCAC − +At, Ptr 158 2 miR158 a UCCCAAAUGUAGACAAAGCA + − At PPR 159 bCCCCAAAUGUAGACAAAGCA − − At 160 3 miR159 a UUUGGAUUGAAGGGAGCUCUA + +At, Gm, Hv*,   MYB 161 Lj, Mt, Os,  Pg*, Ptr, So*,  Sb*, Ta*, Vv,  ZmmiR159 b UUUGGAUUGAAGGGAGCUCUU − + At 162 miR159 c UUUGGAUUGAAGGGAGCUCCU− − At 163 miR319 a-b UUGGACUGAAGGGAGCUCCCU + + At, Bo, Gm,   TCP 164Lt, Os, Ptr,  Ta miR319 c UUGGACUGAAGGGAGCUCCUU − − At, Os 165 4 mir160a-c UGCCUGGCUCCCUGUAUGCCA + + At, Gm, Os,   ARF 166 Ptr, Tt, Zm 5miR161.1 a UUGAAAGUGACUACAUCGGGG + + At PPR 167 miR161.2 aUCAAUGCAUUGAAAGUGACUA + + At 168 6 miR162 a-b UCGAUAAACCUCUGCAUCCAG + +At, Gm, Ll,   DCL 169 Mt, Os, Ptr,  Vv 7 miR163 aUUGAAGAGGACUUGGAACUUCGAU + − At SAMT 170 8 miR164 a-bUGGAGAAGCAGGGCACGUGCA − + At, Pb, Ta NAC 171 miR164 cUGGAGAAGCAGGGCACGUGCG + + At 172 9 miR165 a-b UCGGACCAGGCUUCAUCCCCC − +At, Hc, Ptr HD- 173 ZIPIII miR166 a-g UCGGACCAGGCUUCAUUCCCC + +At, Gm, Hv,   174 In*, Mt, Os,  Ptr, Sb, Zm 10 miR167 a-bUGAAGCUGCCAGCAUGAUCUA + + At, Gm, Os,   ARF 175 Pc*, Ptr, Zm miR167 cUUAAGCUGCCAGCAUGAUCUU − − At 176 miR167 d UGAAGCUGCCAGCAUGAUCUGG + +At, Gm, In,   177 Ptr, So 11 miR168 a-b UCGCUUGGUGCAGGUCGGGAA + +At, Bp, Gm,   AGO1 178 Ht, Hv, Le,   Os, Ptr, Sb, So, St, Vv, Zm 12miR169 a CAGCCAAGGAUGACUUGCCGA + + At, Gm, Os,   HAP2 179 Ptr, PtrmiR169 b-c CAGCCAAGGAUGACUUGCCGG + + At, Gm, Os,   180 Ptr, Zm miR169d-g UGAGCCAAGGAUGACUUGCCG + + At, Ptr 181 miR169 h-nUAGCCAAGGAUGACUUGCCUG + + At, Ls, Os,   182 Pb, Ptr, Sb,   So, Ta 13miR170 a UGAUUGAGCCGUGUCAAUAUC − + At SCR 183 miR171 aUGAUUGAGCCGCGCCAAUAUC + + At, Os, Ptr,   184 Ta, Zm miR171.2 b-cUUGAGCCGUGCCAAUAUCACG + − At, Os, Ptr,   185 Ta, Zm miR171.1 cUGAUUGAGCCGUGCCAAUAUC − + At, Gm, Hc,   186 Hv, Os, Ptr,   Ta, Zm 14miR172 a-b AGAAUCUUGAUGAUGCUGCAU − + At, Gm, Le,   AP2 187 Os, Ptr, StmiR172 c-d AGAAUCUUGAUGAUGCUGCAG + − At, Cs 188 miR172 eGGAAUCUUGAUGAUGCUGCAU − + At, Os, Ptr 189 15 miR173 aUUCGCUUGCAGAGAGAAAUCAC − + At TAS1, 190 TAS2 16 miR390 a-bAAGCUCAGGAGGGAUAGCGCC + + At, Os, Ptr,   TAS3 143 St, Zm miR391 aUUCGCAGGAGAGAUAGCGCCA − + At 144 17 miR393 a-b UCCAAAGGGAUCGCAUUGAUC − −At, Os, Ptr T1R1/ 191 F-box bHLH 18 miR394 a-b UUUGGCAUUCUGUCCACCUCC − −At, Gm, Os,   F-box 192 Ptr, Rp 19 miR395 a,d- CUGAAGUGUUUGGGGGAACUC − −At, Gm, Os,   ATPS 193 e Ptr, Ta miR395 b- CUGAAGUGUUUGGGGGGACUC − − At194 c, f 20 miR396 a UUCCACAGCUUUCUUGAACUG − + At, Bv, Gm,   GRF 195Mc, Os, Ptr,   Ppe, Ptr, So,   St, Zm miR396 b UUCCACAGCUUUCUUGAACUU − −At, Bn, Gm,   196 Mc, Os, Ptr,  St 21 miR397 a UCAUUGAGUGCAGCGUUGAUG − +At, Hv, Os,  laccase 197 Ptr miR397 b UCAUUGAGUGCAUCGUUGAUG − − At 19822 miR398 a UGUGUUCUCAGGUCACCCCUU − − At, Cs, Gm,   CSD 199 Lj, Mt, Os, Ptr miR398 b-c UGUGUUCUCAGGUCACCCCUG − + At, Gm, Ha,   CytC 200Ls, Mt, Nb,   Os, Zm* 23 miR399 a UGCCAAAGGAGAUUUGCCCUG − − At E2- 201UBC miR399 b, c UGCCAAAGGAGAGUUGCCCUG − + At, Mt, Os,   202 Ptr, SbmiR399 d UGCCAAAGGAGAUUUGCCCCG − − At, Os 203 miR399 eUGCCAAAGGAGAUUUGCCUCG − − At 204 miR399 f UGCCAAAGGAGAUUUGCCCGG − −At, Os 205 24 miR403 a aUUAGAUUCACGCACAAACUCG + − At, Ptr AGO2 145 25miR447 a-b UUGGGGACGAGAUGUUUUGUUG − + At 2PGK 146 miR447 cUUGGGGACGACAUCUUUUGUUG − − 206 ^(a)miRNAs are grouped by relatedfamilies, with differences among families underlined; ^(b)Col-0libraries included Col-0 seedling, aerial, and inflorescence tissues,plus jaw-d sequences, rdr2/dcl3 contained seedling libraries from bothmutants, and inflorescence tissues of rdr2; ^(c)Presence of miRNA ingenomic sequence is indicated in regular text, EST sequences are inbold, see information available on the World Wide Web atsanger.ac.ul/Software/Rfam/mirna/index.shtml for primary stem sequences;sequences with 1-2 base changes from the Arabidopsis sequence areindicated by an asterisk.Arabidopsis miRNA Precursors Exhibit Characteristics of Pol IITranscripts

To determine if a reference set of Arabidopsis thaliana MIRNA genetranscripts contain 5′ cap structures typical of RNA pol II transcripts,a series of RNA ligase-mediated 5′RACE reactions were done usingpoly(A)⁺-selected RNA that was pretreated with either calf intestinephosphatase plus tobacco acid pyrophosphatase (CIP+TAP) or buffer alone.Only transcripts containing a 5′ cap should ligate to adapters, andsubsequently amplify by PCR, following CIP+TAP treatment. Transcriptslacking a cap should ligate and amplify only from the sample treatedwith buffer alone. As controls, capped Scarecrow-like6-IV (SCL6-IV,At4g00150) transcript and miR171-guided 3′ cleavage product from SCL6-IV(containing a 5′ monophosphate) were analyzed using gene specific primersets (FIG. 9A) (Llave et al., Science 297:2053-2056, 2002).CIP+TAP-dependent 5′RACE products of the predicted size, ˜400 and ˜1,110bp, were detected using 5′-proximal and cleavage site-proximal primersets, respectively (FIG. 9B, lanes 2 and 4). Buffer-dependent 5′RACEproduct was detected only using the cleavage site-proximal primer set(FIG. 9B, lanes 1 and 3). Using locus-specific primer sets for MIR163,MIR397b and MIR398c, CIP+TAP-dependent products but not buffer-dependentproducts were detected (FIG. 9B, lanes 5-10), indicating that the 5′ endof each miRNA transcript was capped. For 47 out of the 92 ArabidopsisMIRNA loci tested, 5′RACE products from poly(A)⁺-selected and 5′ cappedRNA were detected (see below and Table 8). Combined with previous datafor MIR172b and MIR163, and the evidence for a poly(A) tail on miRNAprecursor transcripts, plant MIRNA genes are likely transcribed by anRNA pol II mechanism. These data are also consistent with recentanalyses of MIRNA gene transcripts from animals (Bracht et al., RNA10:1586-1594, 2004; Cai et al., RNA 10:1957-1966, 2004; Lee et al., EMBOJ. 23:4051-4060, 2004).

Identification of a Core Promoter Element for Arabidopsis MIRNA Genes

Products of 5′RACE reactions were detected using locus-specific primersfor 52 of 99 MIRNA genes tested. Transcription start sites were inferredby sequence analysis of the cloned PCR products. At several loci, suchas MIR171a, MIR172b, and MIR172e, multiple 5′RACE products were detectedand up to three clusters of alternative transcription start sites wereidentified (Table 8).

TABLE 8AValidated miRNA sequences cloned from Arabidopsis small RNA librariesASRP Times database Position miRNA isolated No Locus Chrom. Start EndSequence miR156 233 1423 a 2 10683613 106683632 UGACAGAAGAGAGUGAGCACSEQ ID NO: 155 b 4 15074951  15074970 c 4 15415497  15415516 d 5 3456714   3456733 e 5  3867214   3867233 f 5  9136129   9136148 miR156  3 1662 d 5  3456714   3456734 UUGACAGAAGAGAGUGAGCAC SEQ ID NO: 207miR156   1 1783 e 5  3867213   3867233 GUGACAGAAGAGAGUGAGCACSEQ ID NO: 208 f 5  9136128   9136148 miR156   1 1950 a 2 10683612106683632 UGACAGAAGAGAGUGAGCAC SEQ ID NO: 155 b 4 15074951  15074971 c 415415496  15415516 d 5  3456713   3456733 e 5  3867214   3867234 f 5 9136129   9136149 miR157   1 1424 a 1 24916958  24916939UGACAGAAGAUAGAGAGCAC SEQ ID NO: 209 b 1 24924768  24924787 c 3  6244698  6244679 d 1 18030676  18030657 miR157   6 1770 a 1 24916959  24916939UUGACAGAAGAUAGAGAGCAC SEQ ID NO: 158 b 1 24924767  24924787 c 3  6244699  6244679 miR157   2 1952 d 1 18030677  18030657 CUGACAGAAGAUAGAGAGCACSEQ ID NO: 210 miR157*   1 1782 a 1 24916888  24916868GCUCUCUAGCCUUCUGUCAUC SEQ ID NO: 211 b 1 24924838  24924858 miR158  18 142 a 3  3366373   3366354 UCCCAAAUGUAGACAAAGCA SEQ ID NO: 159 miR158*  1 1727 a 3  3366396   3366416 CUUUGUCUACAAUUUUGGAAA SEQ ID NO: 212miR158*   1 1735 a 3  3366397   3366416 CUUUGUCUACAAUUUUGGAASEQ ID NO: 213 miR158*   1 2007 a 3  3366395   3366416CUUUGUCUACAAUUUUGGAAAA SEQ ID NO: 214 miR159 224 1425 a 1 27716915 27716895 UUUGGAUUGAAGGGAGCUCUA SEQ ID NO: 161 miR159   7 1747 b 1 6220806   6220826 UUUGGAUUGAAGGGAGCUCUU SEQ ID NO: 162 miR159   1 1756b 1  6220804   6220824 UCUUUGGAUUGAAGGGAGCUC SEQ ID NO: 215 a 1 27716917 27716897 miR159   2 1800 a 1 27716915  27716896 UUUGGAUUGAAGGGAGCUCUSEQ ID NO: 216 b 1  6220806   6220825 miR159   1 2011 a 1 27716914 27716895 UUGGAUUGAAGGGAGCUCUA SEQ ID NO: 217 miR319   5 1665 a 412353119  12353139 UUGGACUGAAGGGAGCUCCCU SEQ ID NO: 164 b 5 16677717 16677697 miR160 101 1426 a 2 16347360  16347380 UGCCUGGCUCCCUGUAUGCCASEQ ID NO: 166 b 4  9888999  98889019 c 5 19026405  19026385 miR160   11752 a 2 16347360  16347381 UGCCUGGCUCCCUGUAUGCCAU SEQ ID NO: 218 miR160  1 1754 a 2 16347360  16347381 GCCUGGCUCCCUGUAUGCCA SEQ ID NO: 219 b 4 9888999  98889019 c 5 19026404  19026385 miR160*   1 1941 c 5 19026322 19026342 CGUACAAGGAGUCAAGCAUGA SEQ ID NO: 20 miR161.1   4  111 a 117829398  17829418 UUGAAAGUGACUACAUCGGGG SEQ ID NO: 167 miR161.1   1 497 a 1 17829399  17829418 UGAAAGUGACUACAUCGGGG SEQ ID NO: 221 miR161.1 10 1746 a 1 17829399  17829419 UGAAAGUGACUACAUCGGGGU SEQ ID NO: 222miR161.2 307  563 a 1 17829390  17829410 UCAAUGCAUUGAAAGUGACUASEQ ID NO: 168 miR161.2   6 1707 a 1 17829390  17829411UCAAUGCAUUGAAAGUGACUAC SEQ ID NO: 223 miR161.2   5 1712 a 1 17829390 17829409 UCAAUGCAUUGAAAGUGACU SEQ ID NO: 224 miR161.2   1  213 a 117829391  17829410 CAAUGCAUUGAAAGUGACUA SEQ ID NO: 225 miR162   4  395 a5  2634957   2634937 UCGAUAAACCUCUGCAUCCAG SEQ ID NO: 169 b 5  7740613  7740633 miR163   1 1390 a 1 24888022  24888045 UUGAAGAGGACUUGGAACUUCGAU SEQ ID NO: 170 miR164   2 1427 a 2 19527840  19527860UGGAGAAGCAGGGCACGUGCA SEQ ID NO: 171 b 5   287583    287603 miR164*   21812 c 5  9852751   9852771 CACGUGUUCUACUACUCCAAC SEQ ID NO: 226 miR165 30 1428 a 1    78952     78932 UCGGACCAGGCUUCAUCCCCC SEQ ID NO: 173 b 4  368876    368856 miR166 299  934 a 2 19183311  19183331UCGGACCAGGCUUCAUUCCCC SEQ ID NO: 174 b 3 22933276  22933296 c 5  2838738  2838758 d 5  2840709   2840729 e 5 16792772  16792752 f 5 17533605 17533625 g 5 25522108  25522128 miR166   5 1743 a 2 19183311  19183332UCGGACCAGGCUUCAUUCCCCC SEQ ID NO: 227 b 3 22933276  22933297 c 5 2838738   2838759 d 5  2840709   2840730 miR166   2 1764 a 2 19183310 19183331 UUCGGACCAGGCUUCAUUCCCC SEQ ID NO: 228 miR166   1 1779 a 219183310  19183330 UUCGGACCAGGCUUCAUUCCC SEQ ID NO: 229 miR166*   1 1955a 2 19183198  19183218 GGACUGUUGUCUGGCUCGAGG SEQ ID NO: 230 b 3 22933187 22933207 miR167 160    5 a 3  8108097   8108117 UGAAGCUGCCAGCAUGAUCUASEQ ID NO: 175 b 3 23417152  23417172 miR167   3   35 a 3  8108097  8108116 UGAAGCUGCCAGCAUGAUCU SEQ ID NO: 231 b 3 23417152  23417171 c 111137537  11137556 miR167   2  447 a 3  8108098   8108117GAAGCUGCCAGCAUGAUCUA SEQ ID NO: 232 b 3 23417153  23417172 miR167   2 697 a 3  8108096   8108117 AUGAAGCUGCCAGCAUGAUCUA SEQ ID NO: 233 miR167  5  557 b 3 23417152  23417173 UGAAGCUGCCAGCAUGAUCUAU SEQ ID NO: 234miR167   1  790 b 3 23417151  23417172 GUGAAGCUGCCAGCAUGAUCUASEQ ID NO: 235 miR167   1  281 c 1 11137537  11137557UGAAGCUGCCAGCAUGAUCUG SEQ ID NO: 236 miR167   6  535 c 1 11137537 11137558 UGAAGCUGCCAGCAUGAUCUGG SEQ ID NO: 177 miR168  22 1429 a 410578663  10578683 UCGCUUGGUGCAGGUCGGGAA SEQ ID NO: 178 b 5 18376120 18376100 miR168*   5  489 a 4 10578748  10578768 CCCGCCUUGCAUCAACUGAAUSEQ ID NO: 237 miR168*   1 1970 a 4 10578748  10578767CCCGCCUUGCAUCAACUGAA SEQ ID NO: 238 miR168*   1 2076 a 4 10578747 10578767 UCCCGCCUUGCAUCAACUGAA SEQ ID NO: 239 miR169 614 1430 a 3 4359209   4359189 CAGCCAAGGAUGACUUGCCGA SEQ ID NO: 179 miR169  26 1749a 3  4359209   4359190 CAGCCAAGGAUGACUUGCCG SEQ ID NO: 240 b 5  8527514  8527533 c 5 15888116  15888097 miR169 119 1751 b 5  8527514   8527534CAGCCAAGGAUGACUUGCCGG SEQ ID NO: 180 c 5 15888116  15888096 miR169  121757 a 3  4359211   4359191 UGCAGCCAAGGAUGACUUGCC SEQ ID NO: 241 b 5 8527512   8527532 miR169   4 1762 3  4805824   4805805AGCCAAGGAUGACUUGCCGG SEQ ID NO: 242 4 11483124  11483105 b 5  8527515  8527534 c 5 15888115  15888096 miR169   5 1766 a 3  4359209   4359188CAGCCAAGGAUGACUUGCCGAU SEQ ID NO: 243 miR169   1 1768 a 3  4359210  4359190 GCAGCCAAGGAUGACUUGCCG SEQ ID NO: 244 b 5  8527513   8527533miR169  13 1775 1 20043242  20043223 AGCCAAGGAUGACUUGCCGA SEQ ID NO: 2451 20045256  20045275 a 3  4359208   4359189 miR169   1 1787 1 20043242 20043222 AGCCAAGGAUGACUUGCCGAU SEQ ID NO: 246 1 20045256  20045276 a 3 4359208   4359188 miR169   5 1802 c 5 15888116  15888095CAGCCAAGGAUGACUUGCCGGU SEQ ID NO: 247 miR169   3 1813 b 5  8527515  8527535 AGCCAAGGAUGACUUGCCGGA SEQ ID NO: 248 miR169   1 1817 3 4805804   4805824 AGCCAAGGAUGACUUGCCGGU SEQ ID NO: 249 c 5 15888115 15888095 miR169   2 1820 3  4805803   4805824 AGCCAAGGAUGACUUGCCGGUUSEQ ID NO: 250 miR169   1 1824 b 5  8527514   8527535CAGCCAAGGAUGACUUGCCGGA SEQ ID NO: 251 miR169*   1 1772 a 3  4359018  4359037 GGCAAGUUGUCCUUGGCUAC SEQ ID NO: 252 miR169*   1 1773 b 5 8527595   8527616 GGCAAGUUGUCCUUCGGCUACA SEQ ID NO: 253 miR169  22  276d 1 20043244  20043224 UGAGCCAAGGAUGACUUGCCG SEQ ID NO: 181 e 1 20045254 20045274 f 3  4805826   4805806 g 4 11483126  11483106 miR169 402 1514h 1  6695555   6695535 UAGCCAAGGAUGACUUGCCUG SEQ ID NO: 182 i 3  9873362  9873343 j 3  9873739   9873720 k 3  9876931   9876912 l 3  9877296  9877277 m 3  9879575   9879555 n 3  9879947   9879927 miR169   1 1760h 1  6695554   6695535 AGCCAAGGAUGACUUGCCUG SEQ ID NO: 254 i 3  9873362  9873343 j 3  9873739   9873720 k 3  9876931   9876912 l 3  9877296  9877277 m 3  9879574   9879555 n 3  9879946   9879927 miR169  48 1761i 3  9873363   9873342 UAGCCAAGGAUGACUUGCCUGA SEQ ID NO: 255 j 3 9873740   9873719 l 3  9877297   9877276 n 3  9879947   9879926 miR169  1 1765 i 3  9873362   9873342 AGCCAAGGAUGACUUGCCUGA SEQ ID NO: 256 j 3 9873739   9873719 l 3  9877296   9877276 n 3  9879946   9879926 miR169  3 1771 m 3  9879575   9879554 UAGCCAAGGAUGACUUGCCUGU SEQ ID NO: 257miR169   1 1774 h 1  6695556   6695535 GUAGCCAAGGAUGACUUGCCUGSEQ ID NO: 258 i 3  9873364   9873343 j 3  9873741   9873720 k 3 9876933   9876912 m 3  9879576   9879555 n 3  9879948   9879927 miR169  1 1776 i 3  9873363   9873341 UAGCCAAGGAUGACUUGCCUGA C SEQ ID NO: 259miR169   1 1815 3  4644341   4644361 UAGCCAAGGAUGACUUCCCUUSEQ ID NO: 260 miR169   1 1990 h 1  6695555   6695536UAGCCAAGGAUGACUUGCCU SEQ ID NO: 261 i 3  9873363   9873344 j 3  9873740  9873721 k 3  9876932   9876913 l 3  9877297   9877278 m 3  9879575  9879556 n 3  9879947   9879928 miR170   1 1431 a 5 26428840  26428820UGAUUGAGCCGUGUCAAUAUC SEQ ID NO: 183 miR171  34   39 a 3 19084500 19084520 UGAUUGAGCCGCGCCAAUAUC SEQ ID NO: 184 miR171   1  638 a 319084500  19084519 UGAUUGAGCCGCGCCAAUAU SEQ ID NO: 262 miR171.2   1  444b 1  3961387   3961367 UUGAGCCGUGCCAAUAUCACG SEQ ID NO: 185 c 1 22933780 22933760 miR171.1   1 1876 c 1 22933783  22933763 UGAUUGAGCCGUGCCAAUAUCSEQ ID NO: 186 miR172   1  811 c 3  3599817   3599797AGAAUCUUGAUGAUGCUGCAG SEQ ID NO: 188 d 3 20598970  20598990 miR172*   11854 e 5 24005710  24005729 GCAGCACCAUUAAGAUUCAC SEQ ID NO: 263 a 5 1188298   1188279 miR172*   1 2019 a 5  1188298   1188278GCAGCACCAUUAAGAUUCACA SEQ ID NO: 264 e 5 24005710  24005730 miR173   1 886 a 3  8236168   8236189 UUCGCUUGCAGAGAGAAAUCAC SEQ ID NO: 190miR173*   1 2033 a 3  8236234   8236254 UGAUUCUCUGUGUAAGCGAAASEQ ID NO: 265 miR390  89  754 a 2 16069049  16069069AAGCUCAGGAGGGAUAGCGCC SEQ ID NO: 143 b 5 23654187  23654207 miR390  251703 a 2 16069050  16069069 AGCUCAGGAGGGAUAGCGCC SEQ ID NO: 266 b 523654188  23654207 miR390   3 1784 a 2 16069049  16069068AAGCUCAGGAGGGAUAGCGC SEQ ID NO: 267 b 5 23654187  23654206 miR390   31758 a 2 16069051  16069069 GCUCAGGAGGGAUAGCGCC SEQ ID NO: 268 b 523654189  23654207 miR390   2 1972 a 2 16069050  16069070AGCUCAGGAGGGAUAGCGCCA SEQ ID NO: 269 b 5 23654188  23654208 miR390*   1 206 b 5 23654260  23654279 CGCUAUCCAUCCUGAGUUCC SEQ ID NO: 270 miR390*  1 2051 b 5 23654260  23654280 CGCUAUCCAUCCUGAGUUCCA SEQ ID NO: 271miR391   7 1728 a 5 24310386  24310406 UUCGCAGGAGAGAUAGCGCCASEQ ID NO: 144 miR391*   1 1991 a 5 24310737  24310457ACGGUAUCUCUCCUACGUAGC SEQ ID NO: 272 miR396*   1 1724 a 2  4149413  4149434 GGUUCAAUAAAGCUGUGGGAAG SEQ ID NO: 273 miR397   1 1794 a 4 2625958   2625979 UCAUUGAGUGCAGCGUUGAUGU SEQ ID NO: 274 miR398   4 1994b 5  4691110   4391130 UGUGUUCUCAGGUCACCCCUG SEQ ID NO: 200 c 5  4394781  4694801 miR399   1 1867 b 1 23349074  23349054 CCUGCCAAAGGAGAGUUGCCCSEQ ID NO: 275 ASRP1839   3 1839 a 1 29427439  29427458UUCGAUGUCUAGCAGUGCCA SEQ ID NO: 276 miR447   1 1890 a 4  1528188  1528209 UUGGGGACGAGAUGUUUUGUUG SEQ ID NO: 146 b 4  1535480   1535501  0 c 4  1523381   1523360 UUGGGGACGACAUCUUUUGUUG SEQ ID NO: 206 miR403  1  359 a 2 19422223  19422244 AUUAGAUUCACGCACAAACUCG SEQ ID NO: 145

Table 8B provides another summary of locus-specific expression of 99predicted miRNA genes coding for validated Arabidopsis microRNAs.Expression of a specific locus was considered definitive (dark shading)if a primary transcript was detected by 5′ or 3′RACE, or a unique miRNAsequence was cloned or amplified from the ASRP library described here(gray shading with total clones sequenced) or from another publishedlibrary (Other Refs.). The number of clones corresponding to a specificmiRNA or miRNA*(in parentheses) sequence in the ASRP database is shown.Sequences that were detected only in other studies are indicated byorange in the 3′RACE and references columns Loci for which data supportexpression from more than one possible gene are indicated by lightshading.

TABLE 8B Locus specific expression of 99 predicted MIRNA genes codingfor validated Arabidopsis microRNAs

nt, not tested. References cited are: 1. Allen et al., Nat Genet36:1282-1290, 2004; 2. Aukerman & Sakai, Plant Cell 15:2730-2741, 2003;3. Chen, Science 303:2022-2025, 2004; 4. Jones-Rhoades & Bartel, MolCell 14:787-799, 2004; 5. Kurihara & Watanabe, Proc Natl Acad Sci U S A101:12753-12758, 2004; 6. Llave et al., Plant Cell 14:1605-1619, 2002;7. Llave et al., Science 297:2053-2056, 2002; 8. Mette et al., PlantPhysiol 130:6-9, 2002; 9. Palatnik et al., Nature 425:257-263, 2003; 10.Park et al., Curr Biol 12:1484-1495, 2002; 11. Reinhardt et al., GenesDev. 16:1616-1626, 2002; 12. Sunkar and Zhu Plant Cell 16:2001-2019,2004; 13. Arabidopsis EST clones were identified for MIR167d (GenBankaccession AU239920) and MIR168a (H77158).

For each 5′RACE product detected, the transcription start site wasassigned to the most highly represented sequence among six randomlyselected clones. In cases where two clustered sequences were equallyrepresented, the extreme 5′ sequence was assigned as the start site.Following this procedure, the 5′ ends representing 63 transcripts fromthe 52 MIRNA loci were identified (FIG. 10 and Table 8). The vastmajority of transcripts initiated with an adenosine (83%) that waspreceded by a pyrimidine residue (FIG. 10A). Twelve loci yieldedmultiple transcripts that were consistent with alternative start sites.Three transcripts (one from MIR156a and two from MIR172b) containedintrons between the 5′ end and foldback sequence. Each of thesecharacteristics is consistent with transcription by RNA pol II.

To identify conserved motifs flanking the initiation sites at eachmapped locus, a 60-bp genomic segment (−50 to +10 relative to the startsite) was computationally analyzed using BioProspector. An 8-nucleotideTATA box-like sequence was detected upstream from 83% of transcriptionstart sites (FIG. 10B). Using MotifMatcher to scan a broader segment(−200 to +50), the TATA-like sequence was shown to be centered atposition removed from the start site (FIG. 10C). The TATA motif atposition −30 is entirely consistent with TATA motifs for protein-codinggenes (Patikoglou et al., Genes Dev 13:3217-3230, 1999; Shahmuradov etal., Nucleic Acids Res 31:114-117, 2003). We conclude, therefore, thatthese are authentic TATA box sequences within core promoter elements ofMIRNA genes.

Expression of Arabidopsis MIRNA Genes

Despite repeated attempts with multiple primer sets, 5′ start sites weremapped for only about one-half of predicted MIRNA genes (Table 8B). Thismay have been due to either less-than-optimal 5′RACE procedures and lowexpression levels (false negative results) or lack of expression of someloci predicted to be MIRNA genes. It is also possible that some primersets were designed within intron sequences. To develop a morecomprehensive account of Arabidopsis MIRNA genes with validatedexpression data, informatic and experimental approaches were taken. Inthe informatic strategy, the ASRP database was scanned forlocus-specific miRNA or miRNA* (miRNA-complementary species within miRNAduplexes) sequences (Gustafson et al., Nucleic Acid Research33:D637-D640, 2005). Unique miRNA or miRNA* sequences specific toMIR158a, MIR167d, MIR173, MIR391, MIR397a and MIR164c loci were eachrepresented in the database (FIG. 10). In addition, unique miRNAsequences specific to MIR319c, MIR398a, and MIR399f were represented inan independent Arabidopsis small RNA library (Table 8B) (Sunkar & Zhu,Plant Cell 16:2001-2019, 2004). For each of three families (MIR390/391,MIR393, and MIR168) in which negative 5′RACE data were obtained,multiple predicted loci encode an identical miRNA that was detected inat least one small RNA library (Reinhart et al., Genes Dev 16:1616-1626,2002; Jones-Rhoades & Bartel, Mol Cell 14:787-799, 2004; Sunkar & Zhu,Plant Cell 16:2001-2019, 2004; Gustafson et al., Nucleic Acid Research33:D637-D640, 2005). For MIR168a, a locus-specific EST clone (GenBankaccession H77158) exists to confirm expression. For two miRNAs that arerepresented by a single locus (miR173 and miR391), expression wasinferred by cloning or detection of the miRNA sequence. Thus, 5′RACE andunambiguous miRNA cloning/detection support expression of 68 of 99predicted Arabidopsis MIRNA loci.

For the remaining 31 predicted MIRNA genes, locus-specific primers weredesigned to amplify sequences immediately downstream of the precursorfoldback sequence through a 3′ RACE procedure. Positive results wereobtained for five loci.

Example 6 Small RNA Formation in Plants

This example provides a demonstration of the ability to produce novelsiRNAs using engineered ta-siRNA-generating loci. This demonstrationincludes miRNA-dependent formation of novel siRNAs for RNAi againstexogenous and endogenous RNA sequences, and phenotypes associated withsilencing of an endogenous gene (phytoene desaturase, or PDS) using theartificial, engineered cassettes.

Development of Constructs for Wild-Type and Artificial Ta-siRNABiogenesis Assays in N. Benthamiana

The following artificial ta-siRNAs targeting Arabidopsis genes encodingphytoene desaturase (PDS) and PINOID (PID), as well as GFP, weredesigned and expressed using an Arabidopsis thaliana TAS/c-basedconstruct: 35S:TAS1c; 35S:TAS1cGFPd3d4 (SEQ ID NO: 277);35S:TAS1cPDSd3d4 (SEQ ID NO: 278); and 35S:TAS1cPIDd3d4 (SEQ ID NO:279). The ta-siRNA constructs were made in the TAS1c context, as shownin FIG. 11.

The artificial ta-siRNAs were expressed in place of the normal 3′D3(+)and 3′D4(+) positions of TAS1c (positional nomenclature as in Allen etal., Cell 121:207-221, 2005). Artificial ta-siRNA sequences were chosenbased on the principles of the asymmetry rules and presented by Schwarzet al. (Cell 115:199-208, 2003) and Khvorova et al. (Cell 115:209-216,2003). The artificial siRNAs chosen were designed as perfectcomplementary matches to their corresponding target genes, although itis assumed that artificial siRNAs may contain mismatches similar tothose in known miRNA:target duplexes (see Allen et al., Cell121:207-221, 2005, for examples). Each of these constructs contained two21-nt siRNA modules, with the siRNAs designed to target mRNAs for GFP,PDS and PID.

Engineered TAS1c loci were expressed using the CaMV 35S promoter and thenos terminator as regulatory elements. The expressed sequence wasinserted between att sites (positions 55 to 79 and 1106 to 1130 in each)for recombination into a “Gateway” vector. The two consecutive,21-nucleotide engineered ta-siRNAs correspond to nucleotide positions520 to 561 in each of SEQ ID NOs: 277, 278, and 279. Vector sequence isshown in positions 1 to 99 and 1090 to 1185 of each of these sequences;primers used to amplify the cassettes hybridize at positions 96 to 123and 1069 to 1089.

Demonstration of Artificial Ta-siRNA Biogenesis and Activity in N.Benthamiana

Transient ta-siRNA expression assays in Nicotiana benthamiana were doneas in Allen et al. (Cell 121:207-221, 2005). Stable Arabidopsis thalianatransgenic lines were created using the Agrobacterium mediated floraldip method. Transgenic seed from transformed plants was plated onMurashige-Skoog media containing kanamycin (50 μg/ml), and blot assayswere done as described in Allen et al. (Cell 121:207-221, 2005).

Introduction of each construct (35S:TAS1c [which forms wild-type TAS1cta-siRNAs], ³⁵S:TAS1cGFPd3d4, 35S:TAS1cPDSd3d4, and 35S:TAS1cPIDd3d4)into N. benthamiana in a transient assay resulted in miR173-dependentformation of ta-siRNAs (FIG. 12). In the case of 35S:TAS1cGFPd3d4, theartificial ta-siRNA construct was co-expressed with a functional GFPgene. Expression of at least one artificial ta-siRNA was detected in amiR173-dependent manner, by blot assay using each construct (FIG. 12).The GFP gene was silenced by the artificial GFP ta-siRNAs in amiR173-dependent manner (FIG. 12).

A PDS artificial ta-siRNA-generating construct was introduced intowild-type Arabidopsis and rdr6-15 and dcl4-2 (Xie et al., Proc Natl AcadSci USA. 102(36):12984-12989, 2005; Epub 2005 Aug. 29) mutant plants.Both strong and weak loss-of-function PDS phenotypes were detected, butonly in wildtype plants and not in rdr6-15 or dcl4-2 plants (Table 9 andFIG. 13). This indicates that functional artificial ta-siRNAs wereformed through the activity of the normal ta-siRNA pathway.

TABLE 9 Observed phenotype classes of Arabidopsis stable transgeniclines expressing engineered TAS1cPDSd3d4 No Phenotype Weak PhenotypeStrong Phenotype Col-0 (n = 102)  8/102 36/102 58/102 rdr6-15 (n = 291)291/291 — — dcl4-2 (n = 15) 15/15 — —

This disclosure describes the discovery of a new system for RNAi invivo, and provides methods, constructs, and compositions useful forexploiting this discovery. The disclosure further provides myriadinitiator sequences and methods for identifying additional initiatorsequences that are useful in directing in vivo generation of predictable21-mer siRNAs, as well as methods of using constructs containing such aninitiator sequence to mediate RNAi. It will be apparent that the precisedetails of the methods described may be varied or modified withoutdeparting from the spirit of the described invention. We claim all suchmodifications and variations that fall within the scope and spirit ofthe disclosure and the claims below.

We claim:
 1. A method for generating a phased small RNA (phasiRNA) in a plant cell, comprising introducing into the plant cell an effective amount of a microRNA (miRNA).
 2. The method of claim 1, wherein the miRNA is 22-nt in length.
 3. The method of claim 1, wherein the plant cell is in a legume.
 4. The method of claim 3, wherein the legume is selected from the group consisting of M. truncatula, soybeans, peanuts, and common.
 5. The method of claim 1, wherein the plant cell is in a non-legume.
 6. The method of claim 5, wherein the non-legume is selected from the group consisting of corns, rice, wheat, barley, oats, rye, sorghum, sugar cane, grapevine, almonds, apple, peach, sugar beet, tomato, potato, tobacco, cotton, lettuce, sunflower, melons, strawberries, and canola.
 7. The method of claim 1, wherein resistance of the plant to a microbe is reduced.
 8. The method of claim 1, wherein the plant enters a symbiotic interaction with a microbe.
 9. The method of claim 7, wherein the microbe is a rhizobial strain.
 10. The method of claim 8, wherein the microbe is a rhizobial strain.
 11. The method of claim 3, wherein nodulation in the plant is improved.
 12. The method of claim 5, wherein nodulation in the plant is improved. 